You searched for subject:(gene targeting).
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University of Texas Southwestern Medical Center
1.
Ellis, Brian Lee.
Improving Viral Vectors for Gene Targeting in Gene Therapy.
Degree: 2011, University of Texas Southwestern Medical Center
URL: http://hdl.handle.net/2152.5/837 
► Over 10,000 monogenic diseases in the world affect one out of every hundred live births (WHO). Gene targeting is a term that is used describe…
(more)
▼ Over 10,000 monogenic diseases in the world affect one out of every hundred live births (WHO).
Gene targeting is a term that is used describe the manipulation of genetic material, either by adding a
gene in a specific locus, creating a mutation at a specific locus, or correcting a
gene at a specific locus. Here, unless otherwise noted, we will use the term to describe the correction of a
gene with a homologous piece of donor genetic material whereby a mutant
gene that causes monogenic disease is essentially replaced by a wild type copy through homologous recombination. Thus,
gene targeting is inherently safer than classic
gene therapy, where a
gene is randomly introduced into the genome and can cause insertional mutagenesis. Although the rates of homologous recombination are low when simply delivering a donor substrate (1 in a million), creating a deoxyribonucleic acid (DNA) double-stranded break in or around the
gene of interest using a nuclease, increases the rate of
gene targeting 30,000-50,000 fold. The delivery of the nuclease and donor substrate to these cells is one of the major hurdles in achieving this type of therapy. However, for classic
gene therapy there have already been many clinical trials using viral vehicles for
gene delivery. One problem with using a virus for
gene therapy is the low titer associated with some types of virus, in particular, lentivirus. In the first part of this dissertation, this problem is addressed by showing that the addition of caffeine during viral production can increase titer up to 8-fold.
Besides lentivirus, other viruses, like Adeno-associated virus (AAV) have been used in clinical trials. There are nine AAV serotypes, but the most-well characterized is AAV2. Because there are situations where AAV is to be used in cells that cannot be transduced with AAV2, it is essential to know which serotype best infects the desired cell type. The second part of the dissertation describes a comprehensive survey of the ability of AAV1-9 and one engineered serotype to transduce primary and immortalized cells from human, mouse, hamster, and monkey origin. Overall, the results show that AAV1 and AAV6 transduce the most cell types at the highest efficiencies.
Though
gene targeting has been achieved using the homing endonuclease I-Sce in AAV2,
targeting has never been achieved using two zinc-finger nucleases (ZFNs) in any AAV serotype. This is significant because the recognition site for I-Sce is not found in the human genome, while ZFNs are designed to specifically bind in or around a
gene of interest. Based on the results from the AAV survey and the advantage of ZFNs, we created an AAV6 virus that carried the genetic information for both ZFNs and donor substrate for
gene targeting in cells containing a GFP
gene targeting system. We also created an AAV6 virus that carried the donor substrate alone. The third part of this dissertation reveals that dual infection at the optimal multiplicities of infection for both AAV viruses can achieve
targeting efficiencies of ~3%, which is…
Advisors/Committee Members: Porteus, Matthew H..
Subjects/Keywords: Gene Targeting; Gene Therapy; Lentivirus
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APA (6th Edition):
Ellis, B. L. (2011). Improving Viral Vectors for Gene Targeting in Gene Therapy. (Thesis). University of Texas Southwestern Medical Center. Retrieved from http://hdl.handle.net/2152.5/837
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Ellis, Brian Lee. “Improving Viral Vectors for Gene Targeting in Gene Therapy.” 2011. Thesis, University of Texas Southwestern Medical Center. Accessed January 16, 2021.
http://hdl.handle.net/2152.5/837.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Ellis, Brian Lee. “Improving Viral Vectors for Gene Targeting in Gene Therapy.” 2011. Web. 16 Jan 2021.
Vancouver:
Ellis BL. Improving Viral Vectors for Gene Targeting in Gene Therapy. [Internet] [Thesis]. University of Texas Southwestern Medical Center; 2011. [cited 2021 Jan 16].
Available from: http://hdl.handle.net/2152.5/837.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Ellis BL. Improving Viral Vectors for Gene Targeting in Gene Therapy. [Thesis]. University of Texas Southwestern Medical Center; 2011. Available from: http://hdl.handle.net/2152.5/837
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
University of Utah
2.
Morton, John Jason.
Zinc finger nuclease-induced double-strand breaks mediate targeted mutagenesis in Caenorhabditis elegans;.
Degree: PhD, Biochemistry;, 2007, University of Utah
URL: http://content.lib.utah.edu/cdm/singleitem/collection/etd1/id/1121/rec/1477
► Zinc finger nucleases (ZFNs) are chimeric proteins composed of a DNA-binding domain comprised of several tandem Cys2His2 zinc fingers and a nonspecific endonuclease domain from…
(more)
▼ Zinc finger nucleases (ZFNs) are chimeric proteins composed of a DNA-binding domain comprised of several tandem Cys2His2 zinc fingers and a nonspecific endonuclease domain from the Type IIs restriction enzyme FokI. When expressed in the nematode Caenorhabditis elagans, these molecules bind it’s DNA at a sequence specified by the zinc fingers and produces double-strand breaks (DSBs) at this target site in somatic tissues. These breaks activate the nematode DNA repair mechanisms, which attempt to repair the lesions either by homologous recombination (HR), using an unbroken homologous DNA template, or by nonhomologous end joining (NHEF), by which the broken ends are simply rejoined without regard for sequence conservation. The latter process will, at some frequency, produce targeted mutations at the break site. The work present characterizes ZFN-mediated mutagenesis in the somatic tissue of the nematode. Expression of sets of ZFNs, designed to specifically cleave either a synthetic site on an extrachromosomal array or a genomic target, resulted in a spectrum of NHEJ-medicated target site mutations at a frequency of about 20% of each locus. Elimination of the canonical NHEJ pathway in the nematode significantly altered the frequency and types of mutations observed, indicating that this process is fundamental in repairing ZFN-induced DSBs. It is hoped that the information gained from these studies will serve as a guide in the use of ZFNs for germline mutagenesis and gene targeting in the nematode. Use of this technology together with an introduced altered DNA template to direct HR-mediated DSB repair will herald the arrival of a long-sought method of efficient gene targeting of C. elagans.
Subjects/Keywords: Chimeric Proteins; Gene Targeting
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APA (6th Edition):
Morton, J. J. (2007). Zinc finger nuclease-induced double-strand breaks mediate targeted mutagenesis in Caenorhabditis elegans;. (Doctoral Dissertation). University of Utah. Retrieved from http://content.lib.utah.edu/cdm/singleitem/collection/etd1/id/1121/rec/1477
Chicago Manual of Style (16th Edition):
Morton, John Jason. “Zinc finger nuclease-induced double-strand breaks mediate targeted mutagenesis in Caenorhabditis elegans;.” 2007. Doctoral Dissertation, University of Utah. Accessed January 16, 2021.
http://content.lib.utah.edu/cdm/singleitem/collection/etd1/id/1121/rec/1477.
MLA Handbook (7th Edition):
Morton, John Jason. “Zinc finger nuclease-induced double-strand breaks mediate targeted mutagenesis in Caenorhabditis elegans;.” 2007. Web. 16 Jan 2021.
Vancouver:
Morton JJ. Zinc finger nuclease-induced double-strand breaks mediate targeted mutagenesis in Caenorhabditis elegans;. [Internet] [Doctoral dissertation]. University of Utah; 2007. [cited 2021 Jan 16].
Available from: http://content.lib.utah.edu/cdm/singleitem/collection/etd1/id/1121/rec/1477.
Council of Science Editors:
Morton JJ. Zinc finger nuclease-induced double-strand breaks mediate targeted mutagenesis in Caenorhabditis elegans;. [Doctoral Dissertation]. University of Utah; 2007. Available from: http://content.lib.utah.edu/cdm/singleitem/collection/etd1/id/1121/rec/1477
University of Texas Southwestern Medical Center
3.
Checketts, Joshua Allen.
Nuclease-Mediated Targeted Gene Insertion at the Adenosine Deaminase Locus in Primary Cells.
Degree: 2013, University of Texas Southwestern Medical Center
URL: http://hdl.handle.net/2152.5/1727
► Gene therapy is the ability to correct diseases at the DNA level and has long been a goal of science and medicine. The earliest gene…
(more)
▼ Gene therapy is the ability to correct diseases at the DNA level and has long been a goal of science and medicine. The earliest
gene therapy clinical trial was for a patient with severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA) deficiency. Initial trials looked promising and the technique was extended to other forms of primary immunodeficiency. Unfortunately, some of the patients enrolled in these trials using retroviral vectors to carry replacement genes resulted in insertional oncogenesis. To avoid the insertional oncogenesis caused by random integration into the genome, we postulated that targeted insertion of the
gene of interest through homologous recombination would prove to be a safer alternative to random viral insertion of a
gene. To this end, we developed several pairs of TAL effector nucleases (TALENs) designed to target exon 1 of ADA. These TALENs function as dimers, and each pair creates a different targeted double strand break near the start site of the ADA
gene. The most effective pair induces a DNA double strand break immediately preceding the ADA start codon. Targeted activity of these TALENs was measured through determining the percent of alleles that undergo mutagenic non-homologous end joining upon exposure to the TALENs, with up to 14% of alleles undergoing such mutations. In order to stimulate
gene targeting at the ADA locus in human cells, these TALENs were nucleofected into the cells as plasmid DNA, along with a donor plasmid that contains the DNA to be inserted flanked by 800bp arms of homology to the cut site. These TALENs were able to stimulate site-specific integration of the desired fragment at rates of up to 10% in human cell lines. Successful targeted
gene insertion was verified through maintained fluorescence, western blots, and sequencing of the targeted alleles through PCR amplification. We demonstrated the ability to enrich for targeted cells through the expression of a selectable marker within the DNA cassette integrated at the ADA locus. In addition to the editing of cell lines, we showed successful stimulation of
gene targeting in patient-derived fibroblasts in 1.5% of cells. We demonstrated the feasibility of using the ADA locus as a safe harbor through the targeted insertion of three therapeutically interesting genes. Finally, we demonstrated the successful targeted
gene insertion in human CD34+ in up to 0.5% of cells treated. The successful
targeting of human CD34+ is especially relevant, as these cells will need to undergo
gene targeting in order to be therapeutically relevant as a curative therapy for SCID due to ADA deficiency.
Advisors/Committee Members: Albanesi, Joseph P., Porteus, Matthew H., Burma, Sandeep, Abrams, John M., Sternweis, Paul C..
Subjects/Keywords: Gene Therapy; Gene Targeting; Severe Combined Immunodeficiency
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APA (6th Edition):
Checketts, J. A. (2013). Nuclease-Mediated Targeted Gene Insertion at the Adenosine Deaminase Locus in Primary Cells. (Thesis). University of Texas Southwestern Medical Center. Retrieved from http://hdl.handle.net/2152.5/1727
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Checketts, Joshua Allen. “Nuclease-Mediated Targeted Gene Insertion at the Adenosine Deaminase Locus in Primary Cells.” 2013. Thesis, University of Texas Southwestern Medical Center. Accessed January 16, 2021.
http://hdl.handle.net/2152.5/1727.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Checketts, Joshua Allen. “Nuclease-Mediated Targeted Gene Insertion at the Adenosine Deaminase Locus in Primary Cells.” 2013. Web. 16 Jan 2021.
Vancouver:
Checketts JA. Nuclease-Mediated Targeted Gene Insertion at the Adenosine Deaminase Locus in Primary Cells. [Internet] [Thesis]. University of Texas Southwestern Medical Center; 2013. [cited 2021 Jan 16].
Available from: http://hdl.handle.net/2152.5/1727.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Checketts JA. Nuclease-Mediated Targeted Gene Insertion at the Adenosine Deaminase Locus in Primary Cells. [Thesis]. University of Texas Southwestern Medical Center; 2013. Available from: http://hdl.handle.net/2152.5/1727
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Georgia Tech
4.
Ruff, Patrick.
Protein-assisted targeting of genes in yeast and human cells.
Degree: PhD, Biology, 2013, Georgia Tech
URL: http://hdl.handle.net/1853/52907
► This work was designed as a proof-of-principle concept or prototype to show the effect of protein-assisted targeting of DNA to specific genomic loci. Two strategies…
(more)
▼ This work was designed as a proof-of-principle concept or prototype to show the effect of protein-assisted
targeting of DNA to specific genomic loci. Two strategies were employed to deliver the DNA with the aim that once inside the cell the DNA would be delivered to the target sequence by the assistance of a protein. In our case, the chosen protein was the site-specific meganuclease I-SceI. The first strategy described herein was to bind the
targeting DNA to I-SceI by the use of a fusion protein between I-SceI and a known DNA-binding domain, the GAL4-DBD. The second strategy involved using a DNA aptamer to I-SceI to link the
targeting DNA and I-SceI. Testing in vivo revealed that in our human cells (HEK-293) single-stranded DNA was more efficient at
gene targeting than double-stranded DNA. In order for the first strategy to work, we needed to have some region of double-stranded DNA. We found that in human cells, it was better for
gene targeting to have that double-stranded DNA on the 5’ side of our
targeting DNA. We also used gel shift assays to confirm binding by our candidate DNA-binding domain, the GAL4-DBD. We were unable to detect expression of the fusion protein of I-SceI and the GAL4-DBD. For the second strategy we were able to construct an aptamer to I-SceI using a variant of the systematic evolution of ligands by exponential enrichment (SELEX). The I-SceI aptamer was synthesized as part of a longer DNA molecule containing homology to a target locus. Using this chimeric oligonucleotide (part aptamer, part DNA repair region) testing was done in both yeast and human cells. Aside from instances where the aptamer’s secondary structure may have been compromised, the aptamer containing oligonucleotide stimulated repair at a rate 2 to 15-fold higher than the non-selected control sequence. These experimental results show that by delivering
targeting DNA within close proximity to the site of modification,
gene targeting frequencies can be increased.
Advisors/Committee Members: Storici, Francesca (advisor), Chernoff, Yury (committee member), Lieberman, Raquel (committee member), Lobachev, Kirill (committee member), Fan, Yuhong (committee member).
Subjects/Keywords: Aptamer; SELEX; I-SceI; Gene targeting; Protein-assisted targeting
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APA (6th Edition):
Ruff, P. (2013). Protein-assisted targeting of genes in yeast and human cells. (Doctoral Dissertation). Georgia Tech. Retrieved from http://hdl.handle.net/1853/52907
Chicago Manual of Style (16th Edition):
Ruff, Patrick. “Protein-assisted targeting of genes in yeast and human cells.” 2013. Doctoral Dissertation, Georgia Tech. Accessed January 16, 2021.
http://hdl.handle.net/1853/52907.
MLA Handbook (7th Edition):
Ruff, Patrick. “Protein-assisted targeting of genes in yeast and human cells.” 2013. Web. 16 Jan 2021.
Vancouver:
Ruff P. Protein-assisted targeting of genes in yeast and human cells. [Internet] [Doctoral dissertation]. Georgia Tech; 2013. [cited 2021 Jan 16].
Available from: http://hdl.handle.net/1853/52907.
Council of Science Editors:
Ruff P. Protein-assisted targeting of genes in yeast and human cells. [Doctoral Dissertation]. Georgia Tech; 2013. Available from: http://hdl.handle.net/1853/52907
University of Hong Kong
5.
左妍.
Gene targeting to study a
novel acrosome specific gene VAD1.3/AEP1 in
spermatogenesis.
Degree: 2008, University of Hong Kong
URL: http://hdl.handle.net/10722/54422
Subjects/Keywords: Spermatogenesis.; Gene
targeting.
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APA ·
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APA (6th Edition):
左妍. (2008). Gene targeting to study a
novel acrosome specific gene VAD1.3/AEP1 in
spermatogenesis. (Thesis). University of Hong Kong. Retrieved from http://hdl.handle.net/10722/54422
Note: this citation may be lacking information needed for this citation format:
Author name may be incomplete
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
左妍. “Gene targeting to study a
novel acrosome specific gene VAD1.3/AEP1 in
spermatogenesis.” 2008. Thesis, University of Hong Kong. Accessed January 16, 2021.
http://hdl.handle.net/10722/54422.
Note: this citation may be lacking information needed for this citation format:
Author name may be incomplete
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
左妍. “Gene targeting to study a
novel acrosome specific gene VAD1.3/AEP1 in
spermatogenesis.” 2008. Web. 16 Jan 2021.
Note: this citation may be lacking information needed for this citation format:
Author name may be incomplete
Vancouver:
左妍. Gene targeting to study a
novel acrosome specific gene VAD1.3/AEP1 in
spermatogenesis. [Internet] [Thesis]. University of Hong Kong; 2008. [cited 2021 Jan 16].
Available from: http://hdl.handle.net/10722/54422.
Note: this citation may be lacking information needed for this citation format:
Author name may be incomplete
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
左妍. Gene targeting to study a
novel acrosome specific gene VAD1.3/AEP1 in
spermatogenesis. [Thesis]. University of Hong Kong; 2008. Available from: http://hdl.handle.net/10722/54422
Note: this citation may be lacking information needed for this citation format:
Author name may be incomplete
Not specified: Masters Thesis or Doctoral Dissertation
University of Toronto
6.
Nelson, Seana ML.
Generating C-peptide Humanized Mice.
Degree: 2012, University of Toronto
URL: http://hdl.handle.net/1807/69989
► Type 1 diabetes is primarily caused by the loss of insulin producing beta-cells. Future therapies based on transplantation of in vitro generated beta-cells hold great…
(more)
▼ Type 1 diabetes is primarily caused by the loss of insulin producing beta-cells. Future therapies based on transplantation of in vitro generated beta-cells hold great promise for providing a cure. The development of such treatments heavily relies on the use of mouse models. However, insulin expressed from transplanted, mouse derived beta-cells cannot be differentiated from the host in allograft studies. ELISA can distinguish mouse and human C-peptide, a biomarker of insulin secretion. This project aims to create a humanized C-peptide mouse line that could be used to distinguish insulin production in allograft transplantation studies, or differentiate expression from the two mouse insulin genes. Humanized C-peptide insulin 1 chimeras have been generated and an insulin 2 founder has been identified. Following germline transmission, the lines will be crossed to obtain double homozygous humanized C-peptide mice. These animals will be an invaluable resource in allogeneic beta-cell transplants and in monitoring insulin allele expression.
MAST
Advisors/Committee Members: Nagy, Andras, Medical Science.
Subjects/Keywords: Mouse model; Gene targeting; 0369; 0307
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APA (6th Edition):
Nelson, S. M. (2012). Generating C-peptide Humanized Mice. (Masters Thesis). University of Toronto. Retrieved from http://hdl.handle.net/1807/69989
Chicago Manual of Style (16th Edition):
Nelson, Seana ML. “Generating C-peptide Humanized Mice.” 2012. Masters Thesis, University of Toronto. Accessed January 16, 2021.
http://hdl.handle.net/1807/69989.
MLA Handbook (7th Edition):
Nelson, Seana ML. “Generating C-peptide Humanized Mice.” 2012. Web. 16 Jan 2021.
Vancouver:
Nelson SM. Generating C-peptide Humanized Mice. [Internet] [Masters thesis]. University of Toronto; 2012. [cited 2021 Jan 16].
Available from: http://hdl.handle.net/1807/69989.
Council of Science Editors:
Nelson SM. Generating C-peptide Humanized Mice. [Masters Thesis]. University of Toronto; 2012. Available from: http://hdl.handle.net/1807/69989
Hong Kong University of Science and Technology
7.
Guo, Weilin LIFS.
Utilization of non-homologous end joining (NHEJ) mediated knock-in method via CRISPR/Cas9 system to achieve visible conditional knock-out in zebrafish.
Degree: 2016, Hong Kong University of Science and Technology
URL: http://repository.ust.hk/ir/Record/1783.1-95287
;
https://doi.org/10.14711/thesis-b1610645
;
http://repository.ust.hk/ir/bitstream/1783.1-95287/1/th_redirect.html
► In biological studies, insertion of exogenous sequences, such as protein tags, fluorescent reporters or loxp sites into targeted genomic locus provides a powerful tool to…
(more)
▼ In biological studies, insertion of exogenous sequences, such as protein tags, fluorescent reporters or loxp sites into targeted genomic locus provides a powerful tool to investigate the spatial-temporal expression patterns of a gene and its functions of interest. To achieve this goal in zebrafish, the engineered endonucleases (EENs), such as TALENs or CRISPR/Cas9 are utilized, as the first step to generate double strand breaks (DSBs). Subsequently, while majority of these DSBs are repaired by non-homology end joining (NHEJ) pathway that generates small deletion or insertion, a small number of them, if provided with donor plasmid containing homologous arms, could also be repaired by homologous recombination (HR) that results in the precise replacement of endogenous DNA fragment by the sequence of interest. Since HR-mediated knock-in methods occur at such a low frequency (~1%), the CRISPR/Cas9 system has been further modified to break the genomic locus and donor plasmid simultaneously. This concurrent cleavage eventually leads to the direct ligation of genomic DNA and donor sequence through non-homology end joining (NHEJ) pathway at high efficiency (20~40%). Based on these principles for developing knock-in methods, it’s further modified to achieve visible conditional knock-out in zebrafish. Targeting at Tyrosine hydroxylase (th) for its high efficient sgRNA target, high expression level and identifiable expression pattern, the newly developed visible conditional knock-out methods were proved feasible for further studying target genes expression patterns and their functions in temporal-spatial resolution.
Subjects/Keywords: Genetic engineering
; Gene targeting
; Zebra danio
; Genetics
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APA (6th Edition):
Guo, W. L. (2016). Utilization of non-homologous end joining (NHEJ) mediated knock-in method via CRISPR/Cas9 system to achieve visible conditional knock-out in zebrafish. (Thesis). Hong Kong University of Science and Technology. Retrieved from http://repository.ust.hk/ir/Record/1783.1-95287 ; https://doi.org/10.14711/thesis-b1610645 ; http://repository.ust.hk/ir/bitstream/1783.1-95287/1/th_redirect.html
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Guo, Weilin LIFS. “Utilization of non-homologous end joining (NHEJ) mediated knock-in method via CRISPR/Cas9 system to achieve visible conditional knock-out in zebrafish.” 2016. Thesis, Hong Kong University of Science and Technology. Accessed January 16, 2021.
http://repository.ust.hk/ir/Record/1783.1-95287 ; https://doi.org/10.14711/thesis-b1610645 ; http://repository.ust.hk/ir/bitstream/1783.1-95287/1/th_redirect.html.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Guo, Weilin LIFS. “Utilization of non-homologous end joining (NHEJ) mediated knock-in method via CRISPR/Cas9 system to achieve visible conditional knock-out in zebrafish.” 2016. Web. 16 Jan 2021.
Vancouver:
Guo WL. Utilization of non-homologous end joining (NHEJ) mediated knock-in method via CRISPR/Cas9 system to achieve visible conditional knock-out in zebrafish. [Internet] [Thesis]. Hong Kong University of Science and Technology; 2016. [cited 2021 Jan 16].
Available from: http://repository.ust.hk/ir/Record/1783.1-95287 ; https://doi.org/10.14711/thesis-b1610645 ; http://repository.ust.hk/ir/bitstream/1783.1-95287/1/th_redirect.html.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Guo WL. Utilization of non-homologous end joining (NHEJ) mediated knock-in method via CRISPR/Cas9 system to achieve visible conditional knock-out in zebrafish. [Thesis]. Hong Kong University of Science and Technology; 2016. Available from: http://repository.ust.hk/ir/Record/1783.1-95287 ; https://doi.org/10.14711/thesis-b1610645 ; http://repository.ust.hk/ir/bitstream/1783.1-95287/1/th_redirect.html
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
University of Notre Dame
8.
Zhi Xu.
Dissection of the Multiple Functions of Plasminogen
Activator Inhibitor-1</h1>.
Degree: Chemistry and Biochemistry, 2008, University of Notre Dame
URL: https://curate.nd.edu/show/9w032229q44
► Plasminogen Activator Inhibitor-1 (PAI-1) is the main physiological regulator of tissue-type plasminogen activator (tPA) in normal plasma. In addition to its critical function in…
(more)
▼ Plasminogen Activator Inhibitor-1 (PAI-1) is
the main physiological regulator of tissue-type plasminogen
activator (tPA) in normal plasma. In addition to its critical
function in fibrinolysis, PAI-1 has been implicated in other
physiological and pathophysiological processes. Interestingly, both
antifibrinolytic-related and non-antifibrinolytic-related roles of
PAI-1 function have been implicated in the process of angiogenesis,
which might account for some discrepancies observed in angiogenic
models using PAI-1 deficient and over-expressing transgenic mice.
To investigate the structure-function relationships of mouse PAI-1,
the recombinant PAI-1 proteins were expressed in Escherichia coli
and characterized. Our studies indicated that the complex
interactions traditionally associated with different functions of
human PAI-1 apply to the murine system, thus demonstrating a
commonality of subtle functions among different species and
evolutionary conservation of this protein. In an effort to separate
these functions in vivo, knock-in
targeting vector with targeted
mutations to ablate the vitronectin binding ability was
constructed. The composition of the
targeting vector includes the
5’ and 3’ flanking sequences containing the mutation, a
LoxP-FLT-Neo-FLT-LoxP cassette, and a CDA expression cassette.
After homologous recombination with the WT allele, the “floxed”
cassette may be removed by crossing with either Cre recombinase or
flipase expressing transgenic mice. A cell culture-free system was
developed to examine the cre-recombinase-mediated excision of the
“floxed” element before the
targeting vector was introduced into
the embryonic stem cells. In order to bypass the lengthy
backcrossing process from the 129 background into the C57BL/6
background, the homologous arms in the
targeting vector were
constructed using a C57BL/6 genetic background to facilitate the
backcrossing. The
targeting vectors were introduced into both C57
and C57/129 hybrid embryonic stem cells by electroporation, and the
homologous recombination was screened by Southern Blot analysis.
Positive ES cell clones with homologous recombination at both arms
were identified. These PAI-1 knock-in mice would serve to further
elucidate mechanisms associated with the observed phenotypes in
PAI-1 deficient mice in a number of challenge
models.
Advisors/Committee Members: Dr. Francis J. Castellino, Committee Member, Dr. Holly Goodson, Committee Member, Dr. Jeffrey S.Schorey, Committee Member, Dr. Dinshaw S Balsara , Committee Chair, Dr. Anthony S. Serianni, Committee Member.
Subjects/Keywords: coagulation; gene targeting
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APA (6th Edition):
Xu, Z. (2008). Dissection of the Multiple Functions of Plasminogen
Activator Inhibitor-1</h1>. (Thesis). University of Notre Dame. Retrieved from https://curate.nd.edu/show/9w032229q44
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Xu, Zhi. “Dissection of the Multiple Functions of Plasminogen
Activator Inhibitor-1</h1>.” 2008. Thesis, University of Notre Dame. Accessed January 16, 2021.
https://curate.nd.edu/show/9w032229q44.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Xu, Zhi. “Dissection of the Multiple Functions of Plasminogen
Activator Inhibitor-1</h1>.” 2008. Web. 16 Jan 2021.
Vancouver:
Xu Z. Dissection of the Multiple Functions of Plasminogen
Activator Inhibitor-1</h1>. [Internet] [Thesis]. University of Notre Dame; 2008. [cited 2021 Jan 16].
Available from: https://curate.nd.edu/show/9w032229q44.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Xu Z. Dissection of the Multiple Functions of Plasminogen
Activator Inhibitor-1</h1>. [Thesis]. University of Notre Dame; 2008. Available from: https://curate.nd.edu/show/9w032229q44
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
University of Southern California
9.
Yaegashi, Junko.
Application of genome-wide strategies for the mining of
secondary metabolite biosynthesis pathways in filamentous
fungi.
Degree: PhD, Pharmaceutical Sciences, 2017, University of Southern California
URL: http://digitallibrary.usc.edu/cdm/compoundobject/collection/p15799coll3/id/553827/rec/854
► Genome projects of filamentous fungi have generated an unprecedented amount of information, and in a moment in time often referred to as the “post-genomic era”,…
(more)
▼ Genome projects of filamentous fungi have generated an
unprecedented amount of information, and in a moment in time often
referred to as the “post-genomic era”, it is up to us to find ways
to provide meaning to this immense quantity of genome sequence
data. Fungi have long been known to be prolific producers of
bioactive secondary metabolites, but bioinformatic analysis has
showed that these organisms harbor the potential to produce far
more secondary metabolites than are currently known. The work
herein describes genome-wide strategies to approach two main
challenges we now face: 1) finding ways to access the hidden
natural products and 2) developing and/ or using genetic systems in
fungal species to link secondary metabolites to their biosynthesis
genes and elucidate their biosynthesis pathways. We have
demonstrated the power of combining bioinformatics, molecular
gene
targeting, and natural product chemistry in secondary metabolite
biosynthesis research. ❧ We initally used A. nidulans as the target
species and took advantage of its well-established
gene targeting
system. First, genome-based deletion analysis in A. nidulans led us
to find genes involved in the biosynthesis of xanthones located in
at least three distinct loci in the genome. This was particularly
interesting because it contradicted the general notion that fungal
secondary metabolites are clustered. This highlighted the utility
of genomics combined with efficient
gene targeting to identify
these dispersed genes. Next, we hypothesized that altering the
expression of kinases would have an effect on secondary metabolite
production and may activate silent
gene clusters. We used a
genome-wide kinase knockout library and screened its secondary
metabolite profile and found that the mpkA deletion strain produced
aspernidine A, a compound not previously isolated from A. nidulans.
We performed additional
gene deletions and determined the border of
the biosynthesis
gene cluster and proposed the biosynthesis pathway
for aspernidine A. ❧ We then took advantage of the recent advances
in genome sequencing of Penicillium species and chose Penicillium
canescens as the target species. This species is a prolific
producer of secondary metabolites but no previous work has been
reported to link their metabolites to biosynthesis genes. This is
due in large part to the lack of an efficient
gene targeting system
in this organism. We therefore developed a genetic system in P.
canescens that would allow us to perform targeted
gene
manipulations rapidly and efficiently. We then applied this system
and generated a genome-wide deletion library of polyketide synthase
(PKS) genes and nonribosomal peptide synthetase (NRPS) genes. Using
this library, we successfully linked four secondary metabolites,
griseofulvin, xanthoepocin, 15-deoxyoxalicine B, and amauromine, to
their core secondary metabolite biosynthesis genes. Interestingly,
15-deoxyoxalicine B is a structurally unique diterpenic
meroterpenoid, a class of fungal metabolites whose biosynthesis
mechanism remains largely unknown.…
Advisors/Committee Members: Wang, Clay C.C. (Committee Chair), Stiles, Bangyan L. (Committee Member), Olenyuk, Bogdan (Committee Member), Okamoto, Curtis Toshio (Committee Member), Zhang, Chao (Committee Member).
Subjects/Keywords: Aspergillus; Penicillium; genome mining; gene targeting
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APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Yaegashi, J. (2017). Application of genome-wide strategies for the mining of
secondary metabolite biosynthesis pathways in filamentous
fungi. (Doctoral Dissertation). University of Southern California. Retrieved from http://digitallibrary.usc.edu/cdm/compoundobject/collection/p15799coll3/id/553827/rec/854
Chicago Manual of Style (16th Edition):
Yaegashi, Junko. “Application of genome-wide strategies for the mining of
secondary metabolite biosynthesis pathways in filamentous
fungi.” 2017. Doctoral Dissertation, University of Southern California. Accessed January 16, 2021.
http://digitallibrary.usc.edu/cdm/compoundobject/collection/p15799coll3/id/553827/rec/854.
MLA Handbook (7th Edition):
Yaegashi, Junko. “Application of genome-wide strategies for the mining of
secondary metabolite biosynthesis pathways in filamentous
fungi.” 2017. Web. 16 Jan 2021.
Vancouver:
Yaegashi J. Application of genome-wide strategies for the mining of
secondary metabolite biosynthesis pathways in filamentous
fungi. [Internet] [Doctoral dissertation]. University of Southern California; 2017. [cited 2021 Jan 16].
Available from: http://digitallibrary.usc.edu/cdm/compoundobject/collection/p15799coll3/id/553827/rec/854.
Council of Science Editors:
Yaegashi J. Application of genome-wide strategies for the mining of
secondary metabolite biosynthesis pathways in filamentous
fungi. [Doctoral Dissertation]. University of Southern California; 2017. Available from: http://digitallibrary.usc.edu/cdm/compoundobject/collection/p15799coll3/id/553827/rec/854
10.
Pinho, Raquel de Mello e.
Uso do silenciamento gênico mediado por RNA de interferência e de TAL effector nucleases para aumento de eventos gene targeting em células de cão.
Degree: Mestrado, Anatomia dos Animais Domésticos e Silvestres, 2014, University of São Paulo
URL: http://www.teses.usp.br/teses/disponiveis/10/10132/tde-13012015-102643/
;
► A inserção de DNA exógeno no genoma hospedeiro é conseguida principalmente através da utilização de vias de reparo como a junção de pontas não homólogas,…
(more)
▼ A inserção de DNA exógeno no genoma hospedeiro é conseguida principalmente através da utilização de vias de reparo como a junção de pontas não homólogas, que possui caráter aleatório, e a recombinação homóloga, que possibilita o gene targeting. Algumas ferramentas como as TAL Effector Nucleases (TALENs) e o RNA interferência (RNAi) podem ser utilizadas para aumentar a taxa de integração específica e assim melhorar a eficiência e o direcionamento da edição gênica. Nesse trabalho utilizamos o silenciamento gênico mediano por short interference RNA (siRNA) para inibição temporária dos genes ATF7IP uma metiltrasferase, EP300 uma acetiltransferase e KU70 (NHEJ) e um par de TALENs complementares a uma região do gene da distrofina canina. Células Caninas MDCK I foram transfectadas por lipofectamina 2000 (Invitrogen) com 320pmol de siRNAs para ATF7IP e Ep300; e 64 pmol do SiRNA para KU70 em diferentes grupos, 40 horas depois as células foram transfectadas com 15 μg vetor molde derivado do pEGFP-N1 (Clonatech) e com 10 μg dos RNAm das TALENs. A seleção se deu em meio DMEM high com 600μg/ mL de G418 (Lonza) por 14-16 dias. As colônias coletadas através de biópsias foram analisadas por Polimerase Chain Reaction e sequenciamento gênico. Três pares de primers foram utilizados; um controle endógeno (GAPDH), um controle interno do inserto (Neo qPCR) e um para confirmação da recombinação homóloga (DMD3). Os grupos apresentaram grande variação na taxa de mortalidade celular e consequentemente no número de colônias: Com o grupo ATF7IP+Vetor (648c) apresentando maior número de colônias e o grupo EP300+Ku70+Vetor+TALENs o menor (1c). A maior taxa de recombinação ocorreu nos grupos no grupo ATF7IP +Ku70+Vetor+TALENs com 40% das células positivas para neomicina apresentado o evento gene targeting, um aumento considerável na taxa de recombinação quando comparada a porcentagem de 3,1% do controle transfectado somente com o vetor molde. Mostrando que o uso conjunto das TALENs com siRNAs foi um sucesso para o aumento de eventos de edição gênica direcionada.
The insertion of exogenous DNA into a host genome is achieved primarily through the use of DNA repair pathways such as Non-Homologous End Joining (NHEJ) and the Homologous Recombination (HR). The integration by NHEJ has a random feature and is much more common than HR insertions, which are more likely to produce gene targeting events . TAL effector nucleases (TALENs) and RNA interference (RNAi) can be used to increase the rate of specific integration and thus improving the efficiency of gene editing. In this work, we used short interference RNA (siRNA)-mediated gene silencing for transient inhibition of genes ATF7IP (implicated in histone methylation), EP300 (acetyltransferase) and Ku70 (essential to NHEJ) and a pair of TALENs RNAm complementary to canine muscle dystrophin (DMD) gene. MDCK I Canine Cells were transfected by lipofectamine 2000 (Invitrogen) with 320 pmol of siRNAs for ATF7IP and EP300; and 64 pmol of siRNA for Ku70 in different groups. After 40 hours cells were…
Advisors/Committee Members: Ambrosio, Carlos Eduardo.
Subjects/Keywords: Gene targeting; Gene targeting; Homologous recombination; Recombinação homóloga; RNAi; RNAi; TALENs; TALENs
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APA ·
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MLA ·
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Manager
APA (6th Edition):
Pinho, R. d. M. e. (2014). Uso do silenciamento gênico mediado por RNA de interferência e de TAL effector nucleases para aumento de eventos gene targeting em células de cão. (Masters Thesis). University of São Paulo. Retrieved from http://www.teses.usp.br/teses/disponiveis/10/10132/tde-13012015-102643/ ;
Chicago Manual of Style (16th Edition):
Pinho, Raquel de Mello e. “Uso do silenciamento gênico mediado por RNA de interferência e de TAL effector nucleases para aumento de eventos gene targeting em células de cão.” 2014. Masters Thesis, University of São Paulo. Accessed January 16, 2021.
http://www.teses.usp.br/teses/disponiveis/10/10132/tde-13012015-102643/ ;.
MLA Handbook (7th Edition):
Pinho, Raquel de Mello e. “Uso do silenciamento gênico mediado por RNA de interferência e de TAL effector nucleases para aumento de eventos gene targeting em células de cão.” 2014. Web. 16 Jan 2021.
Vancouver:
Pinho RdMe. Uso do silenciamento gênico mediado por RNA de interferência e de TAL effector nucleases para aumento de eventos gene targeting em células de cão. [Internet] [Masters thesis]. University of São Paulo; 2014. [cited 2021 Jan 16].
Available from: http://www.teses.usp.br/teses/disponiveis/10/10132/tde-13012015-102643/ ;.
Council of Science Editors:
Pinho RdMe. Uso do silenciamento gênico mediado por RNA de interferência e de TAL effector nucleases para aumento de eventos gene targeting em células de cão. [Masters Thesis]. University of São Paulo; 2014. Available from: http://www.teses.usp.br/teses/disponiveis/10/10132/tde-13012015-102643/ ;
University of Toronto
11.
Seigel, Kyle.
Enhancing the Efficiency of CRISPR/Cas9 Precise Gene Editing for Cystic Fibrosis Gene Therapy.
Degree: 2018, University of Toronto
URL: http://hdl.handle.net/1807/91348
► Cystic fibrosis (CF) is the most common cause of chronic obstructive lung disease in children and young adults, yet there is no cure for this…
(more)
▼ Cystic fibrosis (CF) is the most common cause of chronic obstructive lung disease in children and young adults, yet there is no cure for this disease. Intimations of curative strategies have emerged from gene therapy, but these approaches have failed in clinical trials due to insufficient safety and efficacy profiles. The molecular technology necessary to overcome preeminent challenges in gene therapy may reside in programmable nucleases – namely CRISPR/Cas9 – which allow for site-specific genomic integration of therapeutic transgenes. However, the clinical utility of these technologies is limited by inherently low efficiencies. Here, we use flow cytometry and PCR assays to demonstrate the efficacy of perturbing DNA repair to overcome such limitations. We demonstrate that overexpressing CtIP and EXO1-4D can enhance the efficiency of CRISPR/Cas9-directed transgene integration by ~3- and ~6-fold, respectively. These discoveries may provide necessary impetus for translating gene therapies into clinical realities for genetic diseases such as CF.
M.Sc.
Advisors/Committee Members: Hu, Jim, Laboratory Medicine and Pathobiology.
Subjects/Keywords: CRISPR/Cas9; CtIP; Cystic fibrosis; EXO1; Gene targeting; Gene therapy; 0564
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APA ·
Chicago ·
MLA ·
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CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Seigel, K. (2018). Enhancing the Efficiency of CRISPR/Cas9 Precise Gene Editing for Cystic Fibrosis Gene Therapy. (Masters Thesis). University of Toronto. Retrieved from http://hdl.handle.net/1807/91348
Chicago Manual of Style (16th Edition):
Seigel, Kyle. “Enhancing the Efficiency of CRISPR/Cas9 Precise Gene Editing for Cystic Fibrosis Gene Therapy.” 2018. Masters Thesis, University of Toronto. Accessed January 16, 2021.
http://hdl.handle.net/1807/91348.
MLA Handbook (7th Edition):
Seigel, Kyle. “Enhancing the Efficiency of CRISPR/Cas9 Precise Gene Editing for Cystic Fibrosis Gene Therapy.” 2018. Web. 16 Jan 2021.
Vancouver:
Seigel K. Enhancing the Efficiency of CRISPR/Cas9 Precise Gene Editing for Cystic Fibrosis Gene Therapy. [Internet] [Masters thesis]. University of Toronto; 2018. [cited 2021 Jan 16].
Available from: http://hdl.handle.net/1807/91348.
Council of Science Editors:
Seigel K. Enhancing the Efficiency of CRISPR/Cas9 Precise Gene Editing for Cystic Fibrosis Gene Therapy. [Masters Thesis]. University of Toronto; 2018. Available from: http://hdl.handle.net/1807/91348
University of Melbourne
12.
Hassan, Md. Mahmudul.
Developing an improved strategy for gene targeting by homologous recombination for stacking insecticidal genes in Arabidopsis and Brassica crop plants.
Degree: 2018, University of Melbourne
URL: http://hdl.handle.net/11343/213555
► Insect pests are a major factor limiting crop productivity worldwide. While insecticide spraying is an effective means of controlling these pests, this approach also harms…
(more)
▼ Insect pests are a major factor limiting crop productivity worldwide. While insecticide spraying is an effective means of controlling these pests, this approach also harms nonpest insect species such as bees. There is therefore much interest in developing technologies that are more targeted. One such approach is to produce insecticides within the tissues of a crop plant so that only insects that attack these crops will be affected. This PhD study was part of a larger Australia-India Grand Challenge project aimed at generating Brassica crop plants that have resistance to caterpillars and aphids; both major pests species in Australia and India. To develop multiple insect resistant GM Brassica crop plants, the Control of Aphid through RNAi and Bt (CARiB) project aimed to use the Bacillus thuringiensis insecticidal toxin to specifically target pest caterpillars and a plant-expressed dsRNA construct to induce RNA interference in sap-sucking aphids. The first part of this thesis describes how a modified version of the Bacillus thuringiensis insecticidal toxin genes Cry1B and Cry1C was designed to circumvent existing intelecual property (IP). Insecticidal activity of these modified genes was then tested in the model Brassica plant Arabidopsis, which demonstrated that they were effective at preventing leaf damage caused by Diamondback moth caterpillars, a major insect pest of Brassica crops. Subsequent molecular analysis of the transgenic plants confirmed the presence of both Cry1B and Cry1C transcripts, as well as Cry1C protein, indicating that both genes were active in these transgenic lines.
Due to the need to develop the RNAi construct targeting aphids, the CARiB project aimed to generate Brassica plants expressing the Cry genes, and then introduce the RNAi construct into these lines once it became available. To aid rapid integration of these transgenes into elite germplasm, the CARiB project proposed that the two insecticidal traits would be physically linked to the same chromosome using homologous recombination (HR). Thus, the second part of this thesis describes the design of an HR approach that enables multiple transgenes to be stacked at the same position within a chromosome. This involved modifying an existing HR system for transgene stacking and testing different components of this improved system. Due to time constraints arising from strict deadlines associated with the CARiB project, only limiting testing of the improved HR system in the model Brassica Arabidopsis was possible. Initial results indicated that the HR system requires further modification. Problems encountered with the modified HR approach and suggestions for further modification to the system are discussed.
An important aspect of the improved HR system is the requirement for HR to occur in the ovule following transformation using floral dipping. While female germline transformation works well in Arabidopsis, the technique is not performed routinely in Brassica species. Thus, the final part of thesis describes experiments aimed at developing a…
Subjects/Keywords: gene stacking; homologous recombination; gene targeting; Bt brassica; brassica floral dipping
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APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Hassan, M. M. (2018). Developing an improved strategy for gene targeting by homologous recombination for stacking insecticidal genes in Arabidopsis and Brassica crop plants. (Doctoral Dissertation). University of Melbourne. Retrieved from http://hdl.handle.net/11343/213555
Chicago Manual of Style (16th Edition):
Hassan, Md Mahmudul. “Developing an improved strategy for gene targeting by homologous recombination for stacking insecticidal genes in Arabidopsis and Brassica crop plants.” 2018. Doctoral Dissertation, University of Melbourne. Accessed January 16, 2021.
http://hdl.handle.net/11343/213555.
MLA Handbook (7th Edition):
Hassan, Md Mahmudul. “Developing an improved strategy for gene targeting by homologous recombination for stacking insecticidal genes in Arabidopsis and Brassica crop plants.” 2018. Web. 16 Jan 2021.
Vancouver:
Hassan MM. Developing an improved strategy for gene targeting by homologous recombination for stacking insecticidal genes in Arabidopsis and Brassica crop plants. [Internet] [Doctoral dissertation]. University of Melbourne; 2018. [cited 2021 Jan 16].
Available from: http://hdl.handle.net/11343/213555.
Council of Science Editors:
Hassan MM. Developing an improved strategy for gene targeting by homologous recombination for stacking insecticidal genes in Arabidopsis and Brassica crop plants. [Doctoral Dissertation]. University of Melbourne; 2018. Available from: http://hdl.handle.net/11343/213555
University of Southern California
13.
Lei, Yuning.
Engineering viral vectors for gene and cell
targeting.
Degree: PhD, Chemical Engineering, 2011, University of Southern California
URL: http://digitallibrary.usc.edu/cdm/compoundobject/collection/p15799coll127/id/430755/rec/2372
► Gene therapy is the introduction of functional genes into dysfunctional cells to treat potentially incurable diseases. The technique sounds promising, yet there are many hurdles…
(more)
▼ Gene therapy is the introduction of functional genes
into dysfunctional cells to treat potentially incurable diseases.
The technique sounds promising, yet there are many hurdles must be
overcome to make it a practical mean of medicine. Viral vector
mediated
gene therapy remains one of the most promising
gene
therapy techniques as its efficiency and duration is the highest
among the different
gene delivery vehicles. To further refine the
viral vector to enhance the
gene delivery ability, we designed a
strategy by decoupling binding and fusion ability of envelope
protein into two distinct proteins. By pseudotyping the viral
vectors with both an antibody and a fusogenic molecule, we can
target and transduce specific cell types. The underlining mechanism
of targeted transduction is that the viral vector will bind to the
desirable cell types via the cognate antibody antigen interaction
and further be endocytosised into the endosome. Inside the endosome
compartment, the low pH environment will trigger the conformation
change of the fusogenic molecule which will fuse both the viral and
cellular membrane, resulting in releasing the viral core. To
further enhance the targeted transduction, we focused on
engineering the two parameters on the viral vector. In chapter 2,
we engineered the fusion loop of the fusogenic molecule to enhance
the activity of the protein. We demonstrated that the engineered
fusogenic molecules can enhance targeted transduction many folds.
We further looked at the other parameter, the
targeting antibody in
chapter 3. By designing a single chain antibody, we were able to
constructed our
targeting virus a simplifier viral production
protocol and showed that the viral vector still exhibit targeted
transduction. These two chapters concluded our effort in enhancing
the targeted transduction by genetically engineering the component
displayed on the viral membrane.; The fourth chapter in this report
combines the technology of the zinc finger nuclease and the
baculoviral vector. Currently, one of the drawback of utilizing
integrating viral vector is the possibility of turning on oncogenes
when the viral vectors integrated to undesirable sites. In this
chapter, we utilize zinc finger nuclease to generate double
stranded break and further demonstrate the ability of utilizing
this technique to converting the baculoviral vector into
gene
targeting vector. We showed that our system can targeted transduce
both immortalized cell lines as well as human embryonic stem cells.
Techniques that we developed here can further enhance the safety of
viral
gene therapy while retain the efficiency and duration of the
viral vectors.
Advisors/Committee Members: Wang, Pin (Committee Chair), Shing, Katherine S. (Committee Member), Arnold, Donald B. (Committee Member).
Subjects/Keywords: lentiviral vector; baculoviral vector; CD20 targeting; cell targeting; zinc finger nuclease; gene targeting; human embryonic stem cells; genetic modification
Record Details
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APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Lei, Y. (2011). Engineering viral vectors for gene and cell
targeting. (Doctoral Dissertation). University of Southern California. Retrieved from http://digitallibrary.usc.edu/cdm/compoundobject/collection/p15799coll127/id/430755/rec/2372
Chicago Manual of Style (16th Edition):
Lei, Yuning. “Engineering viral vectors for gene and cell
targeting.” 2011. Doctoral Dissertation, University of Southern California. Accessed January 16, 2021.
http://digitallibrary.usc.edu/cdm/compoundobject/collection/p15799coll127/id/430755/rec/2372.
MLA Handbook (7th Edition):
Lei, Yuning. “Engineering viral vectors for gene and cell
targeting.” 2011. Web. 16 Jan 2021.
Vancouver:
Lei Y. Engineering viral vectors for gene and cell
targeting. [Internet] [Doctoral dissertation]. University of Southern California; 2011. [cited 2021 Jan 16].
Available from: http://digitallibrary.usc.edu/cdm/compoundobject/collection/p15799coll127/id/430755/rec/2372.
Council of Science Editors:
Lei Y. Engineering viral vectors for gene and cell
targeting. [Doctoral Dissertation]. University of Southern California; 2011. Available from: http://digitallibrary.usc.edu/cdm/compoundobject/collection/p15799coll127/id/430755/rec/2372
Michigan State University
14.
Jhan, Jing-Ru.
Studies of improving therapeutic outcomes of breast cancer through development of personalized treatments and characterization of gene interactions.
Degree: 2016, Michigan State University
URL: http://etd.lib.msu.edu/islandora/object/etd:4191
► Thesis Ph. D. Michigan State University. Cell and Molecular Biology 2016
With an understanding of the heterogeneity of breast cancer, patients with luminal or HER2…
(more)
▼ Thesis Ph. D. Michigan State University. Cell and Molecular Biology 2016
With an understanding of the heterogeneity of breast cancer, patients with luminal or HER2 breast cancer have more specific treatment options other than traditional chemotherapy, the standard therapy for triple-negative breast cancer (TNBC) patients. However, the response to current treatments as well as the prognosis have been clinical challenges. In fact, breast cancer consists of more than subtypes routinely used based on gene expression. In addition, gene expression is highly correlated with response to treatment and prognosis. This suggests that the development of personalized treatment with targeted therapy could improve the outcomes, especially for the TNBC subtype. To address this need, I used two approaches, the development of pathway-guided individualized treatment and an understanding of the interactions of potential genes for targeted therapy. Considering the complexity of gene and pathway interactions, the probability of pathway activation was predicted using pathway signatures generated by comparing gene expression differences between cells overexpressing interested genes and those expressing GFP. This approach was validated in two subtypes of mouse mammary tumors from MMTV-Myc mice, and then further validated in human TNBC patient-derived xenografts (PDXs). The inhibition of tumor growth in mouse mammary tumors and the regression of tumors in PDXs were observed. These proof-of-principle experiments demonstrated the flexibility of pathway-guided personalized treatment. Because this approach needs the combination of different targeted therapies, it is necessary to understand the characteristics of these targetedgenes and therapies, such as gene-gene interactions. To meet this demand, I studied the effects of Stat3 in Myc-driven tumors. Here, MMTV-Myc mice with conditional knockout Stat3 mice was generated. I noted that the deletion of Stat3 in MMTV-Myc mice accelerated the tumorigenesis as well as delayed the tumor growth with an alteration in the frequency of histological subtypes. These tumors also had deficient angiogenesis. Unexpectedly, mice with this genotype had lactation deficiencies and the lethality of pups was found.This model shared some of the same effects of loss of Stat3 in other oncogene-induced tumors and also had distinct effects compared with other models. This suggests that the oncogene drivers determine the roles of Stat3, an oncogene or tumor suppressor, and emphasizes again the importance of understanding the pathways and interactions in the development of treatment.In sum, these studies demonstrate the potential of guiding individualized treatments in preclinical platforms using bioinformatics analyses. Combined with other genomic profiles, this approach could offer more complete assessments before being translated to practice. In addition, this could be further applied in adaptive clinical trials through matching with mouse models.
Description based on online resource;
Advisors/Committee Members: Andrechek, Eran R, Chan, Christina, Conrad, Susan, Gallo, Kathleen, Xiao, Hua.
Subjects/Keywords: Breast – Cancer – Treatment; HER-2 gene; Gene targeting; Bioinformatics; Molecular biology; Cellular biology
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APA (6th Edition):
Jhan, J. (2016). Studies of improving therapeutic outcomes of breast cancer through development of personalized treatments and characterization of gene interactions. (Thesis). Michigan State University. Retrieved from http://etd.lib.msu.edu/islandora/object/etd:4191
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Jhan, Jing-Ru. “Studies of improving therapeutic outcomes of breast cancer through development of personalized treatments and characterization of gene interactions.” 2016. Thesis, Michigan State University. Accessed January 16, 2021.
http://etd.lib.msu.edu/islandora/object/etd:4191.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Jhan, Jing-Ru. “Studies of improving therapeutic outcomes of breast cancer through development of personalized treatments and characterization of gene interactions.” 2016. Web. 16 Jan 2021.
Vancouver:
Jhan J. Studies of improving therapeutic outcomes of breast cancer through development of personalized treatments and characterization of gene interactions. [Internet] [Thesis]. Michigan State University; 2016. [cited 2021 Jan 16].
Available from: http://etd.lib.msu.edu/islandora/object/etd:4191.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Jhan J. Studies of improving therapeutic outcomes of breast cancer through development of personalized treatments and characterization of gene interactions. [Thesis]. Michigan State University; 2016. Available from: http://etd.lib.msu.edu/islandora/object/etd:4191
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
15.
Novo, Luís.
Decationized polyplexes for targeted delivery of nucleic acids : from carrier design to in vivo evaluation.
Degree: 2014, Universiteit Utrecht
URL: http://dspace.library.uu.nl:8080/handle/1874/300546
► Gene therapy is considered a promising treatment for current intractable diseases. However, the clinical applicability of gene therapy is highly dependent on the development of…
(more)
▼ Gene therapy is considered a promising treatment for current intractable diseases. However, the clinical applicability of
gene therapy is highly dependent on the development of safe and efficient
gene delivery vectors. So far, viral vectors have been used for clinical applications, but due to severe risks associated with viruses, cationic polymers have been evaluated as alternatives to viral vectors. Cationic polymers can easily form nanosized particles with plasmid DNA (pDNA), named polyplexes, via electrostatic interactions and possess an enormous chemical and structural flexibility. Polycation-based vectors have demonstrated high efficiency in vitro, however, they induce severe toxicity and possess suboptimal efficiencies in vivo, mainly due to their cationic nature, which significantly hampers their clinical applicability. With our work we have developed an alternative to conventional polycation-based polyplexes: decationized polyplexes. Unlike the cationic polymer based systems, decationized polyplexes are formed by hydrophilic and neutral polymers and can be obtained by an innovative 3-step process: polyplex formation by electrostatic interaction between pDNA and a polycationic precursor, structure stabilization by disulfide crosslinking, and finally removal of cationic charge - decationization. Structurally, decationized polyplexes consist of a disulfide-crosslinked poly(hydroxypropyl methacrylamide) (pHPMA) core stably entrapping plasmid DNA (pDNA), surrounded by a shell of poly(ethylene glycol) (PEG). Retention of pDNA in the nanoparticles is exclusively based on physical entrapment given by the disulfide crosslinks, which provides an intracellularly triggered release profile, since disulfides are cleaved under the higher reducing environment present inside the cells. Through our study decationized polyplexes have demonstrated important advantages when compared to their cationic counterparts, such as much lower degree of nonspecific uptake and high degree cell specific uptake when decorated with
targeting moieties as demonstrated by several cell uptake studies. Furthermore, in vitro studies showed lower cellular toxicity and in vivo nanotoxicity studies using a zebrafish model showed remarkable lower teratogenicity and mortality profile from decationized polyplexes. Stability evaluation in biological fluids a high stability for prolonged periods was found. Finally, in an in vivo biodistribution study, using tumor bearing mice, decationized polyplexes have shown greater retention in blood circulation and higher target tissue (tumor) accumulation. Given their important advantages, decationized polyplexes were also investigated and optimized for small interfering RNA (siRNA) delivery purposes, showing that decationized polyplexes can be used as a platform for different
gene delivery modalities. In conclusion, decationzed polyplexes have demonstrated to be an important contribution for the development of safer polymeric
gene delivery systems especially for targeted therapies. Importantly, the requirements for…
Advisors/Committee Members: Hennink, Wim, van Nostrum, Rene, Mastrobattista, Enrico.
Subjects/Keywords: Gene delivery; pDNA; siRNA; polymer; biocompatibility; nanoparticle; targeting; biodistribution; in vivo
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APA (6th Edition):
Novo, L. (2014). Decationized polyplexes for targeted delivery of nucleic acids : from carrier design to in vivo evaluation. (Doctoral Dissertation). Universiteit Utrecht. Retrieved from http://dspace.library.uu.nl:8080/handle/1874/300546
Chicago Manual of Style (16th Edition):
Novo, Luís. “Decationized polyplexes for targeted delivery of nucleic acids : from carrier design to in vivo evaluation.” 2014. Doctoral Dissertation, Universiteit Utrecht. Accessed January 16, 2021.
http://dspace.library.uu.nl:8080/handle/1874/300546.
MLA Handbook (7th Edition):
Novo, Luís. “Decationized polyplexes for targeted delivery of nucleic acids : from carrier design to in vivo evaluation.” 2014. Web. 16 Jan 2021.
Vancouver:
Novo L. Decationized polyplexes for targeted delivery of nucleic acids : from carrier design to in vivo evaluation. [Internet] [Doctoral dissertation]. Universiteit Utrecht; 2014. [cited 2021 Jan 16].
Available from: http://dspace.library.uu.nl:8080/handle/1874/300546.
Council of Science Editors:
Novo L. Decationized polyplexes for targeted delivery of nucleic acids : from carrier design to in vivo evaluation. [Doctoral Dissertation]. Universiteit Utrecht; 2014. Available from: http://dspace.library.uu.nl:8080/handle/1874/300546
16.
Merle, Jacob Andrew.
Evidence for IRES Mediated Translation of Gurken.
Degree: 2014, SUNY College at Fredonia
URL: http://hdl.handle.net/1951/65149
► The gene gurken (grk) in Drosophila melanogaster is required to establish the anterior/posterior and dorsal/ventral axes of the egg. When insulin levels are high such…
(more)
▼ The gene gurken (grk) in Drosophila melanogaster is required to establish the anterior/posterior and dorsal/ventral axes of the egg. When insulin levels are high such as when food is readily available, grk is translated using a common mechanism that requires recognition of the 7-methylguanosine cap at the 5’ end of the RNA. Translation of grk requires Vasa activity which is inhibited through phosphorylation is spindle-B (spn-B) mutants. It was discovered in our lab that Insulin/Insulin-like Signaling (IIS) mutations or inhibition of TOR by rapamycin result in increased grk translation in spn-B mutants thereby overcoming this cap-dependent block. Based on these data, we hypothesize that the grk 5’ UTR contains an Internal Ribosomal Entry Site (IRES). An IRES provides an alternative mechanism of translation that occurs through use of a secondary structure in the 5’ UTR in the mRNA. The utility of the IRES is that it allows for translation of important mRNA’s even in the absence of canonical cap-dependent translation factors. These conditions can arise in nature when insulin levels or nutrient availability is low. The presence of an IRES will be explored in vivo through a comparison of transgenic reporter lines containing different luciferase reporter constructs. These constructs will be used to test the hypothesis that the grk 5’ UTR contains an IRES and examine the effect of nutrient limitation, inhibition of TOR, or IIS mutations on grk translation. To localize the IRES within the GRK 5' UTR selective deletion mutations will be made in secondary structures identified through selective 2’-Hydroxyl Acylation and Primer Extension (SHAPE) analysis. The activity of these reporter lines will be analyzed through assaying these transgenic lines. This will allow for the identification of the presence and location of the IRES within the grk 5’ UTR. Here we present data demonstrating the activity of reporter constructs that have been generated by fusion with the 5' UTR of grk. Additionally we propose a new technique using GRNA chromatography to identify the presence of an IRES as well as important secondary structures needed for IRES function using the same reporter constructs.
Subjects/Keywords: Drosophila melanogaster.;
Nucleotide sequence.;
Biology – Experiments.;
Gene targeting.
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APA (6th Edition):
Merle, J. A. (2014). Evidence for IRES Mediated Translation of Gurken.
(Masters Thesis). SUNY College at Fredonia. Retrieved from http://hdl.handle.net/1951/65149
Chicago Manual of Style (16th Edition):
Merle, Jacob Andrew. “Evidence for IRES Mediated Translation of Gurken.
” 2014. Masters Thesis, SUNY College at Fredonia. Accessed January 16, 2021.
http://hdl.handle.net/1951/65149.
MLA Handbook (7th Edition):
Merle, Jacob Andrew. “Evidence for IRES Mediated Translation of Gurken.
” 2014. Web. 16 Jan 2021.
Vancouver:
Merle JA. Evidence for IRES Mediated Translation of Gurken.
[Internet] [Masters thesis]. SUNY College at Fredonia; 2014. [cited 2021 Jan 16].
Available from: http://hdl.handle.net/1951/65149.
Council of Science Editors:
Merle JA. Evidence for IRES Mediated Translation of Gurken.
[Masters Thesis]. SUNY College at Fredonia; 2014. Available from: http://hdl.handle.net/1951/65149
University of Texas Southwestern Medical Center
17.
Voit, Richard Alexander 1983-.
Generation of HIV-Resistant T-Cells and Correction of the Sickle Cell Mutation by Targeted Genome Engineering.
Degree: 2013, University of Texas Southwestern Medical Center
URL: http://hdl.handle.net/2152.5/3330
► Targeted genome engineering is a powerful method to create specific modifications at chromosomal loci. This technique makes it feasible to precisely alter DNA sequences by…
(more)
▼ Targeted genome engineering is a powerful method to create specific modifications at chromosomal loci. This technique makes it feasible to precisely alter DNA sequences by introducing a specific DNA double-strand break, which is repaired by the natural cellular machinery. These double strand breaks are induced by engineered chimeric nucleases – either zinc finger nucleases (ZFNs) or Tal effector nucleases (TALENs) – and depending on the experimental design, can result in
gene disruption,
gene correction or targeted transgene integration. In this thesis, I present two applications of this approach in the context of two prevalent human diseases, HIV infection and sickle cell disease.
HIV infects CD4+ T-cells by binding to the CD4 receptor and either the CCR5 or CXCR4 co-receptor on the surface of those cells. Previously, ZFNs were described that create
gene specific knockouts of CCR5, protecting cells against CCR5-tropic (R5) HIV, but not against CXCR4-tropic (X4) HIV. I hypothesized that combining ZFN-mediated CCR5 disruption with targeted integration of a cassette of anti-HIV genes would confer higher levels of resistance against R5-tropic virus and also be protective against X4-tropic virus. In a T-cell reporter line, I showed that CCR5 disruption alone conferred 16-fold protection against R5-tropic virus but had no effect against X4-tropic HIV. In contrast, CCR5 disruption, combined with targeted
gene integration into that locus, of the anti-HIV restriction factors human-rhesus hybrid TRIM5α, APOBEC3G D128K and Rev M10 was completely protective against both viral tropisms.
Sickle cell disease is caused by a point mutation in the β-globin
gene, and I sought to correct this mutation by synthesizing TALENs specific for that site. The β-globin TALENs stimulated integration of therapeutic β-globin cDNA in approximately 20% of cells prior to selection. Using FDA-approved drugs to select for modified cells, I showed virtually complete enrichment of targeted cells. Furthermore, I used the β-globin TALENs to target GFP to the β-globin start codon and designed TALENs to target tdTomato to the start codon of the γ-globin
gene, upregulation of which is a goal of sickle cell disease pharmacotherapy. In this way, I developed an endogenous dual promoter reporter system and screened for drugs that preferentially upregulated γ-globin.
Advisors/Committee Members: Goodman, Joel M., Porteus, Matthew H., Sternweis, Paul C., Graff, Jonathan M..
Subjects/Keywords: beta-Globins; Gene Targeting; HIV Infections; T-Lymphocytes
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APA (6th Edition):
Voit, R. A. 1. (2013). Generation of HIV-Resistant T-Cells and Correction of the Sickle Cell Mutation by Targeted Genome Engineering. (Thesis). University of Texas Southwestern Medical Center. Retrieved from http://hdl.handle.net/2152.5/3330
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Voit, Richard Alexander 1983-. “Generation of HIV-Resistant T-Cells and Correction of the Sickle Cell Mutation by Targeted Genome Engineering.” 2013. Thesis, University of Texas Southwestern Medical Center. Accessed January 16, 2021.
http://hdl.handle.net/2152.5/3330.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Voit, Richard Alexander 1983-. “Generation of HIV-Resistant T-Cells and Correction of the Sickle Cell Mutation by Targeted Genome Engineering.” 2013. Web. 16 Jan 2021.
Vancouver:
Voit RA1. Generation of HIV-Resistant T-Cells and Correction of the Sickle Cell Mutation by Targeted Genome Engineering. [Internet] [Thesis]. University of Texas Southwestern Medical Center; 2013. [cited 2021 Jan 16].
Available from: http://hdl.handle.net/2152.5/3330.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Voit RA1. Generation of HIV-Resistant T-Cells and Correction of the Sickle Cell Mutation by Targeted Genome Engineering. [Thesis]. University of Texas Southwestern Medical Center; 2013. Available from: http://hdl.handle.net/2152.5/3330
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
University of Texas Southwestern Medical Center
18.
Borkowski, Robert John.
Identifying Context-Specific Synthetic Lethal miRNA Inhibition in Non-Small Cell Lung Cancer.
Degree: 2013, University of Texas Southwestern Medical Center
URL: http://hdl.handle.net/2152.5/1726
► Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related fatalities in the US. This is due in part to a lack of highly…
(more)
▼ Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related fatalities in the US. This is due in part to a lack of highly effective therapies for advanced cases, and this is of special concern as most NSCLC cases are not diagnosed until they are in an advanced, later stage. Recent successes in developing genotypically-targeted therapies with potency only in a well-defined subpopulation of tumors suggests that identifying targeted therapies for additional common NSCLC genotypes will improve patient survival.
In this study I utilized a library of inhibitors to microRNAs, a class of post-transcriptional
gene regulators, to identify novel synthetic lethal miRNA inhibition:molecular mechanism interactions in NSCLC. I accomplished this by screening a panel of 13 NSCLC and immortalized normal lung epithelium (HBEC) cell lines in two phases to identify miRNA inhibitors with selective toxicity in the NSCLC cell lines that were also benign in an HBEC cell line. Two inhibitors, the miR-92a and miR-1226* inhibitors, met these criteria. I then collected toxicity data in an expanded panel of 29 total cell lines. This expanded toxicity data was used to identify p53 loss as a molecular mechanism correlated with sensitivity to the miR-92a and miR-1226* inhibitors in NSCLC cell lines. This was recapitulated by demonstrating sensitivity after knockdown of p53 in the previously resistant HBEC30KT cell line. I determined that the inhibitors were toxic in a very sequence-specific manner and that they down-regulated the miR-17~92 polycistron. Down-regulation of the polycistron was toxic in a context-specific manner, and the down-regulation of the miR-17~92 cluster in sensitive cell lines mimicked activation of a 1α, 25-dihydroxyvitamin D3 response in NSCLC cell lines in a manner consistent with sensitivity to the miR-92a inhibitor.
The results of this investigation demonstrate that the screening approach utilized in this study was capable of identifying a synthetic lethal miRNA inhibition:molecular mechanism interaction, and that I was then able to use a genetically defined model of the mechanism to identify a relevant mechanism of action for the toxic inhibitors.
Advisors/Committee Members: Scaglioni, Pier Paolo, Pertsemlidis, Alexander, White, Michael A., Cobb, Melanie H., Corey, David R..
Subjects/Keywords: Carcinoma, Non-Small-Cell Lung; MicroRNAs; Gene Targeting
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APA ·
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CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Borkowski, R. J. (2013). Identifying Context-Specific Synthetic Lethal miRNA Inhibition in Non-Small Cell Lung Cancer. (Thesis). University of Texas Southwestern Medical Center. Retrieved from http://hdl.handle.net/2152.5/1726
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Borkowski, Robert John. “Identifying Context-Specific Synthetic Lethal miRNA Inhibition in Non-Small Cell Lung Cancer.” 2013. Thesis, University of Texas Southwestern Medical Center. Accessed January 16, 2021.
http://hdl.handle.net/2152.5/1726.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Borkowski, Robert John. “Identifying Context-Specific Synthetic Lethal miRNA Inhibition in Non-Small Cell Lung Cancer.” 2013. Web. 16 Jan 2021.
Vancouver:
Borkowski RJ. Identifying Context-Specific Synthetic Lethal miRNA Inhibition in Non-Small Cell Lung Cancer. [Internet] [Thesis]. University of Texas Southwestern Medical Center; 2013. [cited 2021 Jan 16].
Available from: http://hdl.handle.net/2152.5/1726.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Borkowski RJ. Identifying Context-Specific Synthetic Lethal miRNA Inhibition in Non-Small Cell Lung Cancer. [Thesis]. University of Texas Southwestern Medical Center; 2013. Available from: http://hdl.handle.net/2152.5/1726
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
University of Texas Southwestern Medical Center
19.
Long, Chengzu.
Prevention of Muscular Dystrophy in Mice by Gene Editing.
Degree: 2014, University of Texas Southwestern Medical Center
URL: http://hdl.handle.net/2152.5/3953
► Duchenne muscular dystrophy (DMD) is an inherited X-linked disease caused by mutations in the gene encoding dystrophin, a protein required for muscle fiber integrity. DMD…
(more)
▼ Duchenne muscular dystrophy (DMD) is an inherited X-linked disease caused by mutations in the
gene encoding dystrophin, a protein required for muscle fiber integrity. DMD is characterized by progressive muscle weakness and a shortened lifespan, often along with breathing and heart complications. There is no effective treatment.
RNA-guided nucleases-mediated genome editing, based on Type II CRISPR/Cas systems, offers a new approach to alter the genome. It can precisely remove a mutation in DNA, allowing the DNA repair mechanisms to replace it with a normal copy of the
gene. The benefit of this over other
gene therapy techniques is that it can permanently correct the 'defect' in a
gene rather than just transiently adding a 'functional' one.
We used CRISPR/Cas9-mediated genome editing to correct the dystrophin
gene (Dmd) mutation in the germline of mdx mice, a model for DMD, and then monitored skeletal muscle and heart structure and function. Genome editing produced genetically mosaic animals containing 2 to 100% correction of the Dmd
gene. Histological analysis of skeletal muscle and heart from these corrected mice showed absence of the dystrophic muscle phenotype and restoration of dystrophin expression. In addition, the degree of muscle phenotypic rescue in mosaic mice exceeded the efficiency of
gene correction, likely reflecting an advantage of the corrected stem cells and their contribution to regenerating muscle.
Our experiments provide proof-of-concept that CRISPR/Cas9-mediated genomic editing can correct a causative germline mutation causing muscular dystrophy in a mouse model and prevent development of several characteristic features of the disease. With rapid technological advances of
gene delivery systems and improvements to the CRISPR/Cas9 editing system, this strategy may allow correction of disease-causing mutations in the muscle tissue or iPSCs (induced pluripotent stem cells) from patients with genetic diseases.
Advisors/Committee Members: Wang, Zhigao, Olson, Eric N., Cobb, Melanie H., Chen, Zhijian J..
Subjects/Keywords: CRISPR-Cas Systems; Dystrophin; Gene Targeting; Muscular Dystrophy, Duchenne
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APA ·
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Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Long, C. (2014). Prevention of Muscular Dystrophy in Mice by Gene Editing. (Thesis). University of Texas Southwestern Medical Center. Retrieved from http://hdl.handle.net/2152.5/3953
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Long, Chengzu. “Prevention of Muscular Dystrophy in Mice by Gene Editing.” 2014. Thesis, University of Texas Southwestern Medical Center. Accessed January 16, 2021.
http://hdl.handle.net/2152.5/3953.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Long, Chengzu. “Prevention of Muscular Dystrophy in Mice by Gene Editing.” 2014. Web. 16 Jan 2021.
Vancouver:
Long C. Prevention of Muscular Dystrophy in Mice by Gene Editing. [Internet] [Thesis]. University of Texas Southwestern Medical Center; 2014. [cited 2021 Jan 16].
Available from: http://hdl.handle.net/2152.5/3953.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Long C. Prevention of Muscular Dystrophy in Mice by Gene Editing. [Thesis]. University of Texas Southwestern Medical Center; 2014. Available from: http://hdl.handle.net/2152.5/3953
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
University of Guelph
20.
Cealic, Iulia.
Role of the Breast Cancer Susceptibility 2 BRC Repeats in Homologous Recombination.
Degree: MS, Department of Molecular and Cellular Biology, 2013, University of Guelph
URL: https://atrium.lib.uoguelph.ca/xmlui/handle/10214/5259
► Homologous recombination (HR) is a faithful mechanism for the repair of double-stranded DNA breaks (DSBs) and plays a critical role in maintaining the integrity of…
(more)
▼ Homologous recombination (HR) is a faithful mechanism for the repair of double-stranded DNA breaks (DSBs) and plays a critical role in maintaining the integrity of genomic DNA. The product of the Breast Cancer Susceptibility 2 (BRCA2)
gene functions as a recombination mediator in HR-directed repair of DSBs. BRCA2 interacts directly with RAD51, the central recombinase of HR, through highly conserved repetitive motifs of 30-40 amino acids, named BRC repeats, and regulates the formation of the RAD51-ssDNA nucleoprotein filament. There is significant variability in the number of BRC repeats among taxa. However, all mammalian BRCA2 orthologs have eight BRC repeats, which display different characteristics in in vitro studies of RAD51-ssDNA nucleoprotein filament. To test the importance of the number of BRC repeats and to evaluate the role of individual BRC repeats in HR, BRCA2 variants bearing different combinations of BRC repeats were generated using BAC-recombineering, expressed in murine hybridoma cells, and assayed for the ability to stimulate HR using a
gene targeting assay. The BRCA2 variant bearing BRC repeats 1 to 4 decreased the efficiency of HR and increased the level of Rad51 protein, whereas the BRCA2 variant bearing BRC repeats 5 to 8 significantly stimulated HR, but had no effect on the level of Rad51. These results supported the hypothesis that BRC repeats are not functionally equivalent, but rather have different, perhaps reinforcing functions in HR.
Advisors/Committee Members: Baker, Mark (advisor).
Subjects/Keywords: BRCA2; BRC repeats; Homologous recombination; Gene targeting; Rad51
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APA (6th Edition):
Cealic, I. (2013). Role of the Breast Cancer Susceptibility 2 BRC Repeats in Homologous Recombination. (Masters Thesis). University of Guelph. Retrieved from https://atrium.lib.uoguelph.ca/xmlui/handle/10214/5259
Chicago Manual of Style (16th Edition):
Cealic, Iulia. “Role of the Breast Cancer Susceptibility 2 BRC Repeats in Homologous Recombination.” 2013. Masters Thesis, University of Guelph. Accessed January 16, 2021.
https://atrium.lib.uoguelph.ca/xmlui/handle/10214/5259.
MLA Handbook (7th Edition):
Cealic, Iulia. “Role of the Breast Cancer Susceptibility 2 BRC Repeats in Homologous Recombination.” 2013. Web. 16 Jan 2021.
Vancouver:
Cealic I. Role of the Breast Cancer Susceptibility 2 BRC Repeats in Homologous Recombination. [Internet] [Masters thesis]. University of Guelph; 2013. [cited 2021 Jan 16].
Available from: https://atrium.lib.uoguelph.ca/xmlui/handle/10214/5259.
Council of Science Editors:
Cealic I. Role of the Breast Cancer Susceptibility 2 BRC Repeats in Homologous Recombination. [Masters Thesis]. University of Guelph; 2013. Available from: https://atrium.lib.uoguelph.ca/xmlui/handle/10214/5259
University of Guelph
21.
Knapp, Jennifer.
Investigating the Role of Rad51 in Mammalian Ectopic Homologous Recombination.
Degree: MS, Department of Molecular and Cellular Biology, 2013, University of Guelph
URL: https://atrium.lib.uoguelph.ca/xmlui/handle/10214/7280
► DNA damage occurs through endogenous and exogenous sources, and can lead to stalled replication forks, genetic disorders, cancer, and cell death. Homologous recombination (HR) is…
(more)
▼ DNA damage occurs through endogenous and exogenous sources, and can lead to stalled replication forks, genetic disorders, cancer, and cell death. Homologous recombination (HR) is a relatively fast and error-free repair pathway for damaged DNA, which can occur through a
gene conversion event or through a crossing-over event with the exchange of genetic material. Homologous recombination occurs most frequently in the G2 phase of the cell cycle and utilizes the sister chromatid as the repair template. When the sister chromatid is unavailable, the homologous chromosome or a homologous sequence in an ectopic location can be used to repair the lesion; the latter of which is referred to as ectopic homologous recombination (EHR). Rad51 is a key protein involved in HR, and to test its role in EHR, variant Rad51 proteins were expressed in murine hybridoma cells. These Rad51 variants were assayed for their effects on EHR. Excess wild-type Rad51 as well as a deficiency of wild-type Rad51 decreased EHR from the background level found in these cell lines. Thus, Rad51 is necessary for EHR, but there may be an optimal amount of Rad51 required for efficient EHR. Expression of the Rad51 catalytic mutants Rad51K133A and Rad51K133R was found to have an inhibitory effect on EHR, as expected based on the loss of ATP binding and ATP hydrolysis, respectively, in these variants. Excess wild-type Rad51 was verified in this study to increase HR via a
gene targeting assay. MMC treatment, but not ionizing radiation, leads to an increase in EHR in the presence of excess wild-type Rad51. Thus, endogenous levels of Rad51 are sufficient to maintain EHR, but in the presence of excess wild-type Rad51, the level of EHR can increase in response to certain DNA damaging agents and in response to
gene targeting.
Advisors/Committee Members: Baker, Mark (advisor).
Subjects/Keywords: Ectopic homologous recombination; Murine hybridoma cells; Rad51; DNA damage; Gene Targeting
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APA ·
Chicago ·
MLA ·
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CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Knapp, J. (2013). Investigating the Role of Rad51 in Mammalian Ectopic Homologous Recombination. (Masters Thesis). University of Guelph. Retrieved from https://atrium.lib.uoguelph.ca/xmlui/handle/10214/7280
Chicago Manual of Style (16th Edition):
Knapp, Jennifer. “Investigating the Role of Rad51 in Mammalian Ectopic Homologous Recombination.” 2013. Masters Thesis, University of Guelph. Accessed January 16, 2021.
https://atrium.lib.uoguelph.ca/xmlui/handle/10214/7280.
MLA Handbook (7th Edition):
Knapp, Jennifer. “Investigating the Role of Rad51 in Mammalian Ectopic Homologous Recombination.” 2013. Web. 16 Jan 2021.
Vancouver:
Knapp J. Investigating the Role of Rad51 in Mammalian Ectopic Homologous Recombination. [Internet] [Masters thesis]. University of Guelph; 2013. [cited 2021 Jan 16].
Available from: https://atrium.lib.uoguelph.ca/xmlui/handle/10214/7280.
Council of Science Editors:
Knapp J. Investigating the Role of Rad51 in Mammalian Ectopic Homologous Recombination. [Masters Thesis]. University of Guelph; 2013. Available from: https://atrium.lib.uoguelph.ca/xmlui/handle/10214/7280
Columbia University
22.
SriRamaratnam, Rohitha.
Application and development of methods towards the target identification of biologically-active small molecules.
Degree: 2011, Columbia University
URL: https://doi.org/10.7916/D8T169HJ
► Small molecules have played an important role in defining the functions and identities of numerous proteins involved in fundamental biological processes as well as pathways…
(more)
▼ Small molecules have played an important role in defining the functions and identities of numerous proteins involved in fundamental biological processes as well as pathways involved in disease. Chemical genetics represents the formalization of this process into a defined field desiring to achieve the breadth and specificity of classical genetics. In order to gain full advantage of a small molecule's ability to perturb the cell for novel or desired phenotypes, a complete understanding of the molecule's mechanism of action must be achieved. Identification of the biological targets of a molecule represents the most direct approach to attaining this knowledge.
In our strategy to find novel mechanisms to target cancers with oncogenic RAS mutations, we have used small molecules to probe specific weaknesses of this cancerous network through synthetic lethal screening. One molecule identified in these screens, RSL3, attracted interest as a candidate for target identification studies because of its potent lethality and potentially unique mechanism of action. We used an affinity chromatography approach to directly isolate binding partners of RSL3 by modifying the molecules structure to incorporate various affinity tags. Through these experiments we ultimately identified a number of interesting candidate targets. Investigations validating these targets suggest that multi-targeted modulation of antioxidant and prostaglandin networks may be a mechanism for selectively killing cancers with oncogenic RAS.
The identification of biological targets of small molecules poses a difficult challenge to the field of forward chemical genetics. Thus, we attempted to optimize a unique method for target identification, the yeast three-hybrid system (Y3H), which detects small molecule-protein interactions through a transcriptional assay in vivo. We created a version of our Y3H system that incorporated a covalent anchor and compared it with the existing state-of-the-art, which uses a high affinity non-covalent anchor. Transcriptional assays indicated our new system was functional, but surprisingly could not improve upon the original Y3H system. These results highlight the complexities of manipulating ligand-receptor interactions in vivo.
Subjects/Keywords: Chemistry; Biology; Molecules; Biochemistry; Biochemical genetics; Gene targeting
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APA (6th Edition):
SriRamaratnam, R. (2011). Application and development of methods towards the target identification of biologically-active small molecules. (Doctoral Dissertation). Columbia University. Retrieved from https://doi.org/10.7916/D8T169HJ
Chicago Manual of Style (16th Edition):
SriRamaratnam, Rohitha. “Application and development of methods towards the target identification of biologically-active small molecules.” 2011. Doctoral Dissertation, Columbia University. Accessed January 16, 2021.
https://doi.org/10.7916/D8T169HJ.
MLA Handbook (7th Edition):
SriRamaratnam, Rohitha. “Application and development of methods towards the target identification of biologically-active small molecules.” 2011. Web. 16 Jan 2021.
Vancouver:
SriRamaratnam R. Application and development of methods towards the target identification of biologically-active small molecules. [Internet] [Doctoral dissertation]. Columbia University; 2011. [cited 2021 Jan 16].
Available from: https://doi.org/10.7916/D8T169HJ.
Council of Science Editors:
SriRamaratnam R. Application and development of methods towards the target identification of biologically-active small molecules. [Doctoral Dissertation]. Columbia University; 2011. Available from: https://doi.org/10.7916/D8T169HJ
University of Manchester
23.
Costello, Ryan.
Towards the analyses of cell lineages using conditional gene alterations.
Degree: PhD, 2016, University of Manchester
URL: https://www.research.manchester.ac.uk/portal/en/theses/towards-the-analyses-of-cell-lineages-using-conditional-gene-alterations(af6e20c3-1dd5-4c46-b14e-e062900b812a).html
;
http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.684836
► The ability to precisely modify the mouse genome is an invaluable tool for any researcher. If an artificial epitope sequence is integrated into target loci…
(more)
▼ The ability to precisely modify the mouse genome is an invaluable tool for any researcher. If an artificial epitope sequence is integrated into target loci in specific cell types, it is possible to generate mice with these cells specifically tagged with the epitope, which can be used for many subsequent studies. Homologous recombination and the Cre/loxP system have been used to generate targeted and conditional transgenic mice, which have provided the basis for many studies into gene function. However, in recent years, improvements in technology have led to the development of RNA and protein based methods of specifically editing DNA sequences at user-selected loci. This thesis aimed to investigate the effectiveness of the novel gene targeting methods TALEN and CRISPR/Cas9. It also aimed to utilize different strains of mice generated using the Cre/loxP system in Trichuris muris, an animal infection model of the human disease Trichuris trichiura. TALENs use a pair of protein-based monomers specific to the sense and anti-sense strand of a target DNA sequence to dimerise a FokI nuclease and initiate a deletion in the genome. As a study into the practical use of this emerging technology TALENs were generated to target Oct-4 (a stem cell marker) in order to integrate an artificial epitope sequence, which could be used for enrichment experiments. The CRISPR/Cas9 is a very efficient RNA-based system used for modifying a target sequence. This system has been utilized to integrate an epitope sequence into the Rosa26 locus, downstream of a floxed STOP codon. This allows for expression of the epitope following the introduction of tissue restricted Cre recombinase. IL-1 is an important cytokine in the immune response towards T.muris. IL-1R1 was conditionally removed in CD4 cells and the role of IL-1 signaling in developing Th1, Th2 and Th17 responses was then studied. Interestingly, IL-1R1fl/fl CD4Cre mice could generate Th1 and Th2 response but showed a reduction in IL-22 and IL-17 production, two key Th17 cytokines. Infected IL-1R1fl/fl CD4Cre mice also displayed increased gut morphology and goblet cell hyperplasia. Therefore, it was concluded that IL-1 signaling from CD4 cells has an important role in host defense and the development of a full Th17 response. It was also shown that removing IL-1R1 in naïve mice had no affect on lymphocyte development. IL-10 is an anti-inflammatory cytokine expressed by gut macrophages, which contributes to homeostatic control of the immune system. IL-10R was specifically removed in the macrophage specific Cre lines LysMCre and also in CX3CR1Cre as a way of comparing the two Cre drivers. The mice were then infected with T.muris and displayed significant inflammation and also failure to expel the worms in the LysMCre model. This suggests a role for this model in future studies of gut macrophages. Clearly, animal models are very important in the study of gene function and also as a method of assessing the application of new technologies such as CRISPR/Cas9. Future work with the artificial epitope…
Subjects/Keywords: 572.8; Gene Targeting; CRISPR; TALEN; IL-1; CD4; Rosa26; Artificial Epitope
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APA ·
Chicago ·
MLA ·
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CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Costello, R. (2016). Towards the analyses of cell lineages using conditional gene alterations. (Doctoral Dissertation). University of Manchester. Retrieved from https://www.research.manchester.ac.uk/portal/en/theses/towards-the-analyses-of-cell-lineages-using-conditional-gene-alterations(af6e20c3-1dd5-4c46-b14e-e062900b812a).html ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.684836
Chicago Manual of Style (16th Edition):
Costello, Ryan. “Towards the analyses of cell lineages using conditional gene alterations.” 2016. Doctoral Dissertation, University of Manchester. Accessed January 16, 2021.
https://www.research.manchester.ac.uk/portal/en/theses/towards-the-analyses-of-cell-lineages-using-conditional-gene-alterations(af6e20c3-1dd5-4c46-b14e-e062900b812a).html ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.684836.
MLA Handbook (7th Edition):
Costello, Ryan. “Towards the analyses of cell lineages using conditional gene alterations.” 2016. Web. 16 Jan 2021.
Vancouver:
Costello R. Towards the analyses of cell lineages using conditional gene alterations. [Internet] [Doctoral dissertation]. University of Manchester; 2016. [cited 2021 Jan 16].
Available from: https://www.research.manchester.ac.uk/portal/en/theses/towards-the-analyses-of-cell-lineages-using-conditional-gene-alterations(af6e20c3-1dd5-4c46-b14e-e062900b812a).html ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.684836.
Council of Science Editors:
Costello R. Towards the analyses of cell lineages using conditional gene alterations. [Doctoral Dissertation]. University of Manchester; 2016. Available from: https://www.research.manchester.ac.uk/portal/en/theses/towards-the-analyses-of-cell-lineages-using-conditional-gene-alterations(af6e20c3-1dd5-4c46-b14e-e062900b812a).html ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.684836
Florida Atlantic University
24.
Helmick, Ericka Elizabeth.
Genetic differentiation among populations of bald eagles, Haliaeetus leucocephalus.
Degree: MS, 2011, Florida Atlantic University
URL: http://purl.flvc.org/FAU/3171395
► Summary: The bald eagle, Haliaeetus leucocephalus, population declined dramatically in the early 20th century reducing the population from tens of thousands of birds within the…
(more)
▼ Summary: The bald eagle, Haliaeetus leucocephalus, population declined dramatically in the early 20th century reducing the population from tens of thousands of birds within the lower 48 states, to <450 pairs of birds, effectively inducing a population bottleneck. The overall population has recovered and was removed from the endangered species list in 2007. This study investigates whether such overall population statistics are appropriate descriptors for this widespread species. I investigated the genetic differentiation between three populations of bald eagles from Alaska, North Florida and Florida Bay using both mitochondrial and nuclear DNA loci to determine whether discrete subpopulations comprise the broad range. Significant FST values, for both mtDNA and microsatellites, were found between both Florida populations and Alaska, but not within Florida populations. Results indicate that there is strong population structure, rejecting the null hypothesis of a panmictic population. Future conservation efforts should focus on subpopulations rather than the overall population.
Electronic reproduction. Boca Raton, Fla., 2011.
Subjects/Keywords: Wildlife conservation; Birds – Molecular genetics; Gene targeting; Developmental biology; Biochemical markers
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APA (6th Edition):
Helmick, E. E. (2011). Genetic differentiation among populations of bald eagles, Haliaeetus leucocephalus. (Masters Thesis). Florida Atlantic University. Retrieved from http://purl.flvc.org/FAU/3171395
Chicago Manual of Style (16th Edition):
Helmick, Ericka Elizabeth. “Genetic differentiation among populations of bald eagles, Haliaeetus leucocephalus.” 2011. Masters Thesis, Florida Atlantic University. Accessed January 16, 2021.
http://purl.flvc.org/FAU/3171395.
MLA Handbook (7th Edition):
Helmick, Ericka Elizabeth. “Genetic differentiation among populations of bald eagles, Haliaeetus leucocephalus.” 2011. Web. 16 Jan 2021.
Vancouver:
Helmick EE. Genetic differentiation among populations of bald eagles, Haliaeetus leucocephalus. [Internet] [Masters thesis]. Florida Atlantic University; 2011. [cited 2021 Jan 16].
Available from: http://purl.flvc.org/FAU/3171395.
Council of Science Editors:
Helmick EE. Genetic differentiation among populations of bald eagles, Haliaeetus leucocephalus. [Masters Thesis]. Florida Atlantic University; 2011. Available from: http://purl.flvc.org/FAU/3171395
University of Minnesota
25.
Levine, Rachel.
Exploring Critical Vehicle Parameters for the Design of Multitargeted Nanoparticles for Cancer Specific Gene Delivery.
Degree: PhD, Chemical Engineering, 2015, University of Minnesota
URL: http://hdl.handle.net/11299/188940
► The greatest obstacle to clinical application of cancer gene therapy is lack of effective delivery tools. Gene delivery vehicles must protect against degradation, avoid immunogenic…
(more)
▼ The greatest obstacle to clinical application of cancer gene therapy is lack of effective delivery tools. Gene delivery vehicles must protect against degradation, avoid immunogenic effects and prevent off target delivery which can cause harmful side effects. The stealth liposomes has greatly improved tumor localization of small molecule drugs and is a promising tool for nucleic acid delivery as the polyethylene glycol coating its surface protects against immune recognition and blood clearance. In this study, DNA was fully encapsulated within in stealth liposomes by complexing the macromolecule with a cationic polymer before encapsulation. Formation methods and material compositions were then investigated for their effects on encapsulation. This technology was translated for protective delivery of siRNA designed for HPV viral gene silencing and cervical cancer treatment. Stealth liposomes encapsulating siRNA were functionalized with a targeting peptide which binds to the alpha6 integrin, a cervical cancer biomarker. It was found that both targeting and polymer complexation before encapsulation were critical components to effective transfection. Varying the siRNA:polymer ratio revealed an optimal concentration for enhanced transfection, but no improvement to internalization, suggesting polymer complexation enhances transfection through an intracellular mechanism. Nanoparticles functionalized with cancer-targeting ligands have shown promise but are still limited by off-tumor binding to healthy tissues that nevertheless express low levels of the molecular target. Targeting two unique cancer biomarkers using dual-targeted heteromultivalent nanoparticles presents a solution to this challenge by requiring overexpression of two separate ligands for localization. In order to guide experimental design, a kinetic model was built to explore how the affinity and valency of dual-ligand liposomes affect the binding and selectivity of delivery to cells with various receptor expression. α5β1 and α6β4 integrin expression levels were then quantified on 20 different cell lines to identify appropriate model cells for in vitro investigation. Dual-targeting heteromultivalent liposomes were synthesized using the alpha6 targeting peptide and an alpha5beta1 targeting peptide. Heteromultivalent liposomes with varying peptide ratios were delivered to cells with varying integrin concentrations. Binding and internalization was then evaluated to understand the effect of valency and avidity on binding kinetics and delivery. Dual-ligand liposomes with equal valencies of each targeting peptide achieved enhanced binding efficiency and selectivity for cells expressing equal and high receptor levels. This liposome formulation was used as a gene delivery vehicle to achieve improved transfection to dual-receptor expressing cells. The insights gained from this study inform rational design of modular heteromultivalent nanoparticles for enhanced specificity to target tissue, for the creation of more effective cancer treatments.
Subjects/Keywords: Dual Targeting; Gene Delivery; Peptide Amphiphile; Stealth Liposomes; Targeted Therapies
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APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Levine, R. (2015). Exploring Critical Vehicle Parameters for the Design of Multitargeted Nanoparticles for Cancer Specific Gene Delivery. (Doctoral Dissertation). University of Minnesota. Retrieved from http://hdl.handle.net/11299/188940
Chicago Manual of Style (16th Edition):
Levine, Rachel. “Exploring Critical Vehicle Parameters for the Design of Multitargeted Nanoparticles for Cancer Specific Gene Delivery.” 2015. Doctoral Dissertation, University of Minnesota. Accessed January 16, 2021.
http://hdl.handle.net/11299/188940.
MLA Handbook (7th Edition):
Levine, Rachel. “Exploring Critical Vehicle Parameters for the Design of Multitargeted Nanoparticles for Cancer Specific Gene Delivery.” 2015. Web. 16 Jan 2021.
Vancouver:
Levine R. Exploring Critical Vehicle Parameters for the Design of Multitargeted Nanoparticles for Cancer Specific Gene Delivery. [Internet] [Doctoral dissertation]. University of Minnesota; 2015. [cited 2021 Jan 16].
Available from: http://hdl.handle.net/11299/188940.
Council of Science Editors:
Levine R. Exploring Critical Vehicle Parameters for the Design of Multitargeted Nanoparticles for Cancer Specific Gene Delivery. [Doctoral Dissertation]. University of Minnesota; 2015. Available from: http://hdl.handle.net/11299/188940
University of Minnesota
26.
Dhande, Yogesh.
Glycopolymers for Targeted Gene Delivery and Genome Editing.
Degree: PhD, Chemical Engineering, 2017, University of Minnesota
URL: http://hdl.handle.net/11299/209051
► Targeted delivery of therapeutics is of great interest to reduce toxicity and immunogenicity of the treatment. In particular, the liver is an ideal target for…
(more)
▼ Targeted delivery of therapeutics is of great interest to reduce toxicity and immunogenicity of the treatment. In particular, the liver is an ideal target for nucleic acid therapeutics due to its large size, regenerative capacity, and the role in producing serum proteins. In this work, N-acetyl-D-galactosamine (GalNAc) ligands clustered into a polymeric architecture were studied for enhanced binding to the asialoglycoprotein receptors (ASGPRs) on hepatocytes. A series of cationic glycopolymers based on this architecture was used to encapsulate plasmids (pDNA) into polymer-pDNA complexes (polyplexes) and deliver them to receptor-specific cells. The GalNAc-derived polyplexes were colloidally stable and showed cell type-specific gene expression in cultured cells. This work demonstrated the versatility of glycopolymers in selective delivery of therapeutics to cells of interest. We sought to further understand the role of such gene-delivery vehicles in genome editing applications using the CRISPR/Cas9 system. Our results show that the gene delivery vehicle can play a role in promoting homology-directed repair over nonhomologous end joining based on its gene delivery properties. The frequency of editing correlates with the fraction of cells expressing Cas9 above a certain threshold and higher expression does not contribute to any gains in editing efficiency. Taken together, these observations suggest that future gene-delivery vehicles aimed for genome editing applications should be designed to deliver only a sufficient amount of DNA but to a large fraction of cells.
Subjects/Keywords: Asialoglycoprotein receptor; Gene delivery; Genome editing; Glycopolymer; Liver targeting; Nanoparticle
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APA ·
Chicago ·
MLA ·
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CSE |
Export
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APA (6th Edition):
Dhande, Y. (2017). Glycopolymers for Targeted Gene Delivery and Genome Editing. (Doctoral Dissertation). University of Minnesota. Retrieved from http://hdl.handle.net/11299/209051
Chicago Manual of Style (16th Edition):
Dhande, Yogesh. “Glycopolymers for Targeted Gene Delivery and Genome Editing.” 2017. Doctoral Dissertation, University of Minnesota. Accessed January 16, 2021.
http://hdl.handle.net/11299/209051.
MLA Handbook (7th Edition):
Dhande, Yogesh. “Glycopolymers for Targeted Gene Delivery and Genome Editing.” 2017. Web. 16 Jan 2021.
Vancouver:
Dhande Y. Glycopolymers for Targeted Gene Delivery and Genome Editing. [Internet] [Doctoral dissertation]. University of Minnesota; 2017. [cited 2021 Jan 16].
Available from: http://hdl.handle.net/11299/209051.
Council of Science Editors:
Dhande Y. Glycopolymers for Targeted Gene Delivery and Genome Editing. [Doctoral Dissertation]. University of Minnesota; 2017. Available from: http://hdl.handle.net/11299/209051
University of Minnesota
27.
Adil, Maroof Mohammad.
A platform for next-generation cancer therapies: multi-targeted nonviral vectors for site-specific gene delivery and expression.
Degree: PhD, Chemical Engineering, 2013, University of Minnesota
URL: http://hdl.handle.net/11299/170877
► Advances in genetics have empowered gene therapy as a cancer treatment, however there are many challenges to delivering genes specifically to target disease sites. Presented…
(more)
▼ Advances in genetics have empowered gene therapy as a cancer treatment, however there are many challenges to delivering genes specifically to target disease sites. Presented here is the development of a new non-viral gene delivery vehicle, consisting of branched polyethyleneimine (bPEI) condensed plasmid DNA polyplexes encapsulated within a PR_b functionalized stealth liposome, for the delivery of genes specifically to &alpha5&beta1 integrin overexpressing cancer cells. This new transfection agent mediated higher gene expression than non-targeted stealth liposomes and unencapsulated polyplexes in tissue culture. In a liver-metastatic colorectal cancer mouse model, PR_b functionalized stealth liposomes outperformed non-targeted stealth liposomes and was able to specifically transfect the tumor site while avoiding healthy tissues. In addition, a comparative investigation of the transfection mechanism of PR_b functionalized nanoparticles, DOTAP/DOPE lipoplexes, bPEI polyplexes and stealth liposomes was carried out in DLD-1 cells. Results demonstrated that PR_b functionalized nanoparticles were optimally balanced for the transfection of DLD-1 cells with high colloidal stability, fast integrin mediated internalization kinetics, caveolae mediated uptake and endosomal escape. To further increase the specificity of gene expression in cancer tissue, a new therapeutic plasmid DNA (pNF-&kappaB-DTA) was developed with expression of Diphtheria toxin fragment-A (DTA) gene regulated by the transcriptional activity of NF-&kappaB, which is a transcription factor upregulated in cancer. The multi-targeted gene delivery system formed by encapsulating pNF-&kappaB-DTA/bPEI polyplexes in PR_b functionalized stealth liposomes showed more specific gene expression in cancer cells versus healthy cells compared to either individually targeted system. Transfecting cancer cells using the multi-targeted gene delivery system resulted in a dose-dependent reduction of cellular protein expression and a dose-dependent increase in cytotoxicity. Our therapeutic delivery system specifically eradicated on average 70% of a variety of cancer cells while minimally affecting healthy cells. Moving forward, the modular nature of our non-viral delivery vehicle design can facilitate targeting novel pairs of extracellular receptors and upregulated transcription factors for applications beyond cancer gene therapy.
Subjects/Keywords: Cancer; Gene delivery; Integrin; Liposomes; NF-kB; Transcriptional targeting; Chemical engineering
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APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Adil, M. M. (2013). A platform for next-generation cancer therapies: multi-targeted nonviral vectors for site-specific gene delivery and expression. (Doctoral Dissertation). University of Minnesota. Retrieved from http://hdl.handle.net/11299/170877
Chicago Manual of Style (16th Edition):
Adil, Maroof Mohammad. “A platform for next-generation cancer therapies: multi-targeted nonviral vectors for site-specific gene delivery and expression.” 2013. Doctoral Dissertation, University of Minnesota. Accessed January 16, 2021.
http://hdl.handle.net/11299/170877.
MLA Handbook (7th Edition):
Adil, Maroof Mohammad. “A platform for next-generation cancer therapies: multi-targeted nonviral vectors for site-specific gene delivery and expression.” 2013. Web. 16 Jan 2021.
Vancouver:
Adil MM. A platform for next-generation cancer therapies: multi-targeted nonviral vectors for site-specific gene delivery and expression. [Internet] [Doctoral dissertation]. University of Minnesota; 2013. [cited 2021 Jan 16].
Available from: http://hdl.handle.net/11299/170877.
Council of Science Editors:
Adil MM. A platform for next-generation cancer therapies: multi-targeted nonviral vectors for site-specific gene delivery and expression. [Doctoral Dissertation]. University of Minnesota; 2013. Available from: http://hdl.handle.net/11299/170877
University of Manchester
28.
Costello, Ryan.
Towards the analyses of cell lineages using conditional
gene alterations.
Degree: 2016, University of Manchester
URL: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:295855
► The ability to precisely modify the mouse genome is an invaluable tool for any researcher. If an artificial epitope sequence is integrated into target loci…
(more)
▼ The ability to precisely modify the mouse genome is
an invaluable tool for any researcher. If an artificial epitope
sequence is integrated into target loci in specific cell types, it
is possible to generate mice with these cells specifically tagged
with the epitope, which can be used for many subsequent studies.
Homologous recombination and the Cre/loxP system have been used to
generate targeted and conditional transgenic mice, which have
provided the basis for many studies into
gene function. However, in
recent years, improvements in technology have led to the
development of RNA and protein based methods of specifically
editing DNA sequences at user-selected loci. This thesis aimed to
investigate the effectiveness of the novel
gene targeting methods
TALEN and CRISPR/Cas9. It also aimed to utilize different strains
of mice generated using the Cre/loxP system in Trichuris muris, an
animal infection model of the human disease Trichuris trichiura.
TALENs use a pair of protein-based monomers specific to the sense
and anti-sense strand of a target DNA sequence to dimerise a FokI
nuclease and initiate a deletion in the genome. As a study into the
practical use of this emerging technology TALENs were generated to
target Oct-4 (a stem cell marker) in order to integrate an
artificial epitope sequence, which could be used for enrichment
experiments. The CRISPR/Cas9 is a very efficient RNA-based system
used for modifying a target sequence. This system has been utilized
to integrate an epitope sequence into the Rosa26 locus, downstream
of a floxed STOP codon. This allows for expression of the epitope
following the introduction of tissue restricted Cre
recombinase.IL-1 is an important cytokine in the immune response
towards T.muris. IL-1R1 was conditionally removed in CD4 cells and
the role of IL-1 signaling in developing Th1, Th2 and Th17
responses was then studied. Interestingly, IL-1R1fl/fl CD4Cre mice
could generate Th1 and Th2 response but showed a reduction in IL-22
and IL-17 production, two key Th17 cytokines. Infected IL-1R1fl/fl
CD4Cre mice also displayed increased gut morphology and goblet cell
hyperplasia. Therefore, it was concluded that IL-1 signaling from
CD4 cells has an important role in host defense and the development
of a full Th17 response. It was also shown that removing IL-1R1 in
naïve mice had no affect on lymphocyte development.IL-10 is an
anti-inflammatory cytokine expressed by gut macrophages, which
contributes to homeostatic control of the immune system. IL-10R was
specifically removed in the macrophage specific Cre lines LysMCre
and also in CX3CR1Cre as a way of comparing the two Cre drivers.
The mice were then infected with T.muris and displayed significant
inflammation and also failure to expel the worms in the LysMCre
model. This suggests a role for this model in future studies of gut
macrophages.Clearly, animal models are very important in the study
of
gene function and also as a method of assessing the application
of new technologies such as CRISPR/Cas9. Future work with the
artificial epitope…
Advisors/Committee Members: TRAVIS, MARK MA, Muller, Werner, Travis, Mark.
Subjects/Keywords: Gene Targeting; CRISPR; TALEN; IL-1; CD4; Rosa26; Artificial Epitope
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Record Details
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Costello, R. (2016). Towards the analyses of cell lineages using conditional
gene alterations. (Doctoral Dissertation). University of Manchester. Retrieved from http://www.manchester.ac.uk/escholar/uk-ac-man-scw:295855
Chicago Manual of Style (16th Edition):
Costello, Ryan. “Towards the analyses of cell lineages using conditional
gene alterations.” 2016. Doctoral Dissertation, University of Manchester. Accessed January 16, 2021.
http://www.manchester.ac.uk/escholar/uk-ac-man-scw:295855.
MLA Handbook (7th Edition):
Costello, Ryan. “Towards the analyses of cell lineages using conditional
gene alterations.” 2016. Web. 16 Jan 2021.
Vancouver:
Costello R. Towards the analyses of cell lineages using conditional
gene alterations. [Internet] [Doctoral dissertation]. University of Manchester; 2016. [cited 2021 Jan 16].
Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:295855.
Council of Science Editors:
Costello R. Towards the analyses of cell lineages using conditional
gene alterations. [Doctoral Dissertation]. University of Manchester; 2016. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:295855
University of New South Wales
29.
Hook, Jeff.
Functional identity of the Mammalian Gamma Tropomyosin Gene.
Degree: Medical Sciences, 2012, University of New South Wales
URL: http://handle.unsw.edu.au/1959.4/52274
;
https://unsworks.unsw.edu.au/fapi/datastream/unsworks:10946/SOURCE01?view=true
► The actin filament system is fundamental to cellular functions including regulation of shape, motility, cytokinesis, intracellular trafficking and tissue organisation. Tropomyosins (Tm) are highly conserved…
(more)
▼ The actin filament system is fundamental to cellular functions including regulation of shape, motility, cytokinesis, intracellular trafficking and tissue organisation. Tropomyosins (Tm) are highly conserved components of actin filaments which differentially regulate filament stability and function. The mammalian Tm family consists of four genes; alphaTm, betaTm, gammaTm and deltaTm (Tpm1-4). Multiple Tm isoforms (>40) are generated by alternative splicing, and the expression of these isoforms is highly regulated with spatial and temporal sorting of Tm isoforms into different cellular compartments. The importance of gammaTm
gene products during mammalian development has not been fully explored. In order to further identify the role of mammalian Tm isoforms, the functional specificity of subsets of gammaTm
gene family products in mice was tested using a series of
gene knockouts. Knockout constructs of the amino-terminal exon 1b and carboxy-terminal exons 9a+9b, exon 9c and exon 9d from the gammaTm
gene were used to assess the viability of ES cells and mice. The deletion of amino-terminal coding exon 1b ablates all eleven known gammaTm
gene cytoskeletal products (Tm5NM1-11) and results in non-viable mice. However, the elimination of four exon 9c-containing isoforms (Tm5NM4,7,8,9) shows no impact on embryo viability whereas the deletion of two exon 9d-containing isoforms (Tm5NM1,2) results in partial lethality. The viability of knockout ES cells in vitro was also compromised as neither exon 1b nor exon 9d homozygous knockout ES cells could be generated. In contrast, homozygous knockout ES cells for exons 9a+9b (Tm5NM3,5,6,8,9,10,11) were viable. Results indicate that exons 9a+9b may be required for ES cells to undergo differentiation and a conditional knockout mouse model of exons 9a+9b is being generated to determine whether these
gene products are required for embryogenesis. Furthermore, sperm lacking cytoskeletal Tm products of the gammaTm
gene show preferential transmission of the deleted allele which may indicate a selective advantage. Since all four Tm genes are expressed in early embryos, ES cells and sperm, it is concluded that isoforms from the gammaTm
gene perform specific functions in ES cell viability and embryogenesis that cannot be compensated by the other Tm genes.
Advisors/Committee Members: Gunning, Peter, Medical Sciences, Faculty of Medicine, UNSW, Kavallaris, Maria, Children's Cancer Institute Australia for Medical Research, Faculty of Medicine, UNSW.
Subjects/Keywords: Knockout mice; Cytoskeleton; Tropomyosin; Gene targeting; Embryo development
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APA (6th Edition):
Hook, J. (2012). Functional identity of the Mammalian Gamma Tropomyosin Gene. (Doctoral Dissertation). University of New South Wales. Retrieved from http://handle.unsw.edu.au/1959.4/52274 ; https://unsworks.unsw.edu.au/fapi/datastream/unsworks:10946/SOURCE01?view=true
Chicago Manual of Style (16th Edition):
Hook, Jeff. “Functional identity of the Mammalian Gamma Tropomyosin Gene.” 2012. Doctoral Dissertation, University of New South Wales. Accessed January 16, 2021.
http://handle.unsw.edu.au/1959.4/52274 ; https://unsworks.unsw.edu.au/fapi/datastream/unsworks:10946/SOURCE01?view=true.
MLA Handbook (7th Edition):
Hook, Jeff. “Functional identity of the Mammalian Gamma Tropomyosin Gene.” 2012. Web. 16 Jan 2021.
Vancouver:
Hook J. Functional identity of the Mammalian Gamma Tropomyosin Gene. [Internet] [Doctoral dissertation]. University of New South Wales; 2012. [cited 2021 Jan 16].
Available from: http://handle.unsw.edu.au/1959.4/52274 ; https://unsworks.unsw.edu.au/fapi/datastream/unsworks:10946/SOURCE01?view=true.
Council of Science Editors:
Hook J. Functional identity of the Mammalian Gamma Tropomyosin Gene. [Doctoral Dissertation]. University of New South Wales; 2012. Available from: http://handle.unsw.edu.au/1959.4/52274 ; https://unsworks.unsw.edu.au/fapi/datastream/unsworks:10946/SOURCE01?view=true
30.
Gras, Jan Cornelis Emile.
A novel technology to target adenovirus vectors: application in cells involved in atherosclerosis.
Degree: 2007, Department of Human and Clinical Genetics, Medicine / Leiden University Medical Center (LUMC), Leiden University
URL: http://hdl.handle.net/1887/12431
► In this thesis a novel technology is described to target adenovirus vectors. Adenovirus vectors are powerful tools to modulate gene expression. The use of these…
(more)
▼ In this thesis a novel technology is described to target adenovirus vectors. Adenovirus vectors are powerful tools to modulate gene expression. The use of these vectors however, is hampered by the fact that many for gene therapy interesting cell types do not, or only at low levels express the CAR receptor, necessary for infection. We developed a linker protein consisting of the virus-binding moiety of CAR genetically fused to the chicken protein avidin. Biotinylated ligands for cell specific receptors are bound to the linker protein via the avidin-biotin interaction. This now targeting protein is used to redirect adenovirus vectors to previously refractory cell types. Using this technology endothelial cell lines as well as primary endothelial cells can by infected at low MOI’s using an biotinylated cyclic RGD peptide. Primary bone marrow derived macrophages and macrophage cell lines are easily infected using a biotinylated dA6dG10 oligo nucleotide ligand. In vivo experiments showed a marked reduction of adenovirus mediated transgene expression by the liver, the organ responsible for virus uptake when unmodified adenovirus vectors are administered, after addition of several different ligands to the virus via the linker protein.
Subjects/Keywords: Adenovirus,,; Atherosclerosis; Gene Therapy; Targeting; Adenovirus,,; Atherosclerosis; Gene Therapy; Targeting
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APA ·
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MLA ·
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APA (6th Edition):
Gras, J. C. E. (2007). A novel technology to target adenovirus vectors: application in cells involved in atherosclerosis. (Doctoral Dissertation). Department of Human and Clinical Genetics, Medicine / Leiden University Medical Center (LUMC), Leiden University. Retrieved from http://hdl.handle.net/1887/12431
Chicago Manual of Style (16th Edition):
Gras, Jan Cornelis Emile. “A novel technology to target adenovirus vectors: application in cells involved in atherosclerosis.” 2007. Doctoral Dissertation, Department of Human and Clinical Genetics, Medicine / Leiden University Medical Center (LUMC), Leiden University. Accessed January 16, 2021.
http://hdl.handle.net/1887/12431.
MLA Handbook (7th Edition):
Gras, Jan Cornelis Emile. “A novel technology to target adenovirus vectors: application in cells involved in atherosclerosis.” 2007. Web. 16 Jan 2021.
Vancouver:
Gras JCE. A novel technology to target adenovirus vectors: application in cells involved in atherosclerosis. [Internet] [Doctoral dissertation]. Department of Human and Clinical Genetics, Medicine / Leiden University Medical Center (LUMC), Leiden University; 2007. [cited 2021 Jan 16].
Available from: http://hdl.handle.net/1887/12431.
Council of Science Editors:
Gras JCE. A novel technology to target adenovirus vectors: application in cells involved in atherosclerosis. [Doctoral Dissertation]. Department of Human and Clinical Genetics, Medicine / Leiden University Medical Center (LUMC), Leiden University; 2007. Available from: http://hdl.handle.net/1887/12431
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Open Access Theses and Dissertations
