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You searched for subject:( episomaalinen plasmidi). Showing records 1 – 2 of 2 total matches.

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Tampere University

1. Marttila, Suvi. Establishment and characterisation of new human induced pluripotent stem cell lines and cardiomyocyte differentiation : a comparative view .

Degree: 2017, Tampere University

Research background and aims. The aim of this study was to establish and characterise iPSC-lines generated with two different methods, as well as to differentiate the created cells into cardiomyocytes, maintaining a comparative view. Since traditional culture conditions include xenogenic and undefined components, also an experiment on establishing and maintaining iPSCs feeder-free was conducted. In addition to studying the reprogramming efficiency, also the expression of pluripotency genes was studied quantitatively at mRNA level. Materials and methods. iPSCs generated from patient fibroblasts were characterised by studying the expression of exogenous and endogenous pluripotency genes by PCR an RT-PCR, staining the cells with pluripotency markers, karyotyping and an embryoid body in vitro -differentiation potential assay, and RT-PCR to detect markers for each germ layer. The cardiomyocyte differentiation was performed in co-culture with END-2 cells. Pluripotency gene expression was also studied with real-time qPCR at passages 3 and 9. Results. All studied iPSC-lines except one Geltrex®-line lost at p. 9 showed successful reprogramming with no qualitative differences between sendai-virally or episomally reprogrammed lines. The lines that were cultured feeder-free stained positive for neural markers, and differentiated, neural precursor-like cells were present at all passages, which was not encountered for MEF-cultured lines. For the two cardiac-differentiated lines, the efficiency of differentiation assessed in two ways showed a more efficient differentiation of the sendai-virally reprogrammed line than the one reprogrammed with episomal plasmids. Gene expression studies showed no significant changes in pluripotency gene expression between lines or passages except for the gene NANOG, the expression of which was lower in the later passage than the earlier passage. The reprogramming efficiencies observed were extremely low, in the range of 0,005-0,017%. Conclusions. Although stem cell research is trying to generate feeder-free and xeno-free methods for iPSC generation and maintenance, the method tested in this thesis did not possess real advantages when compared to the MEF-culturing. The reprogramming efficiencies between feeder-free or MEF-cultured lines derived episomally did not differ. The pluripotency genes were already highly expressed in early passage iPSCs. The differences in pluripotency gene expression between early and late passages were small. Cardiac differentiation was more efficient for sendai-virally reprogrammed line compared to episomally differentiated line. However, more lines would be needed to verify these results. TIIVISTELMÄ Tutkielman tausta ja tavoitteet. Tämän Pro Gradu -työn tarkoituksen oli luoda ja karakterisoida kahdella eri menetelmällä uusia indusoituja pluripotentteja kantasolulinjoja, sekä erilaistaa niitä sydänlihassoluiksi END-2-erilaistusmenetelmällä vertailevalla otteella. Koska perinteiset soluviljelymenetelmät sisältävät eläinperäisiä soluja sekä tuntemattomia…

Subjects/Keywords: induced pluripotent stem cell (iPSC); mouse embryonic feeder (MEF); cardiomyocyte; cardiac differentiation; reprogramming efficiency; differentiation efficiency; episomal plasmid indusoitu pluripotentti kantasolu (iPS-solu); hiiren alkion fibroblasti (MEF); episomaalinen plasmidi; sydänlihassolu; sydänerilaistus; uudelleenohjelmointitehokkuus; erilaistustehokkuus

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APA (6th Edition):

Marttila, S. (2017). Establishment and characterisation of new human induced pluripotent stem cell lines and cardiomyocyte differentiation : a comparative view . (Masters Thesis). Tampere University. Retrieved from https://trepo.tuni.fi/handle/10024/101732

Chicago Manual of Style (16th Edition):

Marttila, Suvi. “Establishment and characterisation of new human induced pluripotent stem cell lines and cardiomyocyte differentiation : a comparative view .” 2017. Masters Thesis, Tampere University. Accessed December 11, 2019. https://trepo.tuni.fi/handle/10024/101732.

MLA Handbook (7th Edition):

Marttila, Suvi. “Establishment and characterisation of new human induced pluripotent stem cell lines and cardiomyocyte differentiation : a comparative view .” 2017. Web. 11 Dec 2019.

Vancouver:

Marttila S. Establishment and characterisation of new human induced pluripotent stem cell lines and cardiomyocyte differentiation : a comparative view . [Internet] [Masters thesis]. Tampere University; 2017. [cited 2019 Dec 11]. Available from: https://trepo.tuni.fi/handle/10024/101732.

Council of Science Editors:

Marttila S. Establishment and characterisation of new human induced pluripotent stem cell lines and cardiomyocyte differentiation : a comparative view . [Masters Thesis]. Tampere University; 2017. Available from: https://trepo.tuni.fi/handle/10024/101732

2. Viljamaa, Matleena. Non-viral gene delivery: Carrier-mediated transfection to retinal pigment epithelial cells and endothelial cells.

Degree: Farmaceutiska fakulteten, 2015, University of Helsinki

Gene therapy involves the delivery of exogenous DNA into the target cells in order to produce therapeutic protein or to correct a genetic defect. The use of cationic liposomes and polymers as carriers of DNA is based on observations that positively charged carriers bind to anionic DNA protecting its premature degradation and facilitating its cellular uptake in transfection. The modification of carriers and the engineering of DNA are proposed to enable efficient and prolonged protein expression after transfection. Gene therapy is a potential treatment for age related macular degeneration (AMD). The dysfunction of retinal pigment epithelial (RPE) cells is assumed to be a significant factor in the development of AMD. The aim of this Master's thesis was to study non-viral gene delivery to RPE cells and endothelial cells using several carrier/DNA combinations. Carriers in this study were DOTAP/DOPE/PS liposomes, methacrylamide based (PDMAEMA) micelles, and anionic lipid coated DNA complexes (LCDCs). The carriers were complexed with episomal plasmid DNA or minicircles using secreted alkaline phosphatase (SEAP) gene as a marker gene. Adult retinal pigment epithelial (ARPE-19) cells, human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE), human embryonic primary RPE cells and endothelial cells (EaHy 926) were used in transfections. In ARPE-19 cells linear PBuA-PDMAEMA -based complexes reached the transfection efficiency of positive control whereas in human primary RPE cells star-like PBuAPDMAEMA -based complexes were the most efficient. In human primary RPE cells, SEAP secretion lasted at least 18 days when PDMAEMA-based micelles complexed with plasmid or minicircle with cytomegalovirus (CMV) promoter were used. High nitrogen/phosphate (n/p) ratios of polyplexes decreased cell viability. DOTAP/DOPE/PS/DNA lipoplexes transfected EaHy cells with high efficiency. In hESC-RPE, lipoplexes also exceeded the transfection efficiency of the positive control and the marker protein secretion lasted ~20 days. Human elongation factor 1a (EF1a) promoter could not prevent transgene silencing. Gene delivery did not succeed with LCDCs in any transfection. According to the results, PBuA-PDMAEMA-polymers and DOTAP/DOPE/PS-liposomes complexed with episomal plasmid or minicircles are potential gene delivery agents for further studies in AMD. More investigation is needed i.e. to confirm the transfection efficiency of the complexes in non-dividing cells.

Geeniterapian tarkoituksena on siirtää kohdesoluihin vierasta DNA:ta tuottamaan terapeuttista proteiinia tai korjaamaan geenivirhe. Positiivisesti varautuneiden lipidi- ja polymeerikantaja-aineiden on todettu muodostavan komplekseja negatiivisesti varautuneen DNA:n kanssa suojellen DNA:ta ennenaikaiselta hajoamiselta ja parantavan DNA:n soluunottoa geeninsiirrossa. Kantajamolekyylien kehittäminen ja DNA:n muokkaaminen voivat mahdollistaa pitkäkestoinen proteiinin tuotannon kohdesoluissa. Geeniterapia on mahdollinen tulevaisuuden hoitomuoto silmänpohjan ikärappeumaan,…

Subjects/Keywords: non-viral transfection; retinal pigment epithelium (RPE); lipoplexes; polyplexes; lipid coated DNA complexes (LCDC); episomal plasmid; minicircle; nonviraalinen geeninsiirto; verkkokalvon pigmenttiepiteeli (RPE); lipopleksi; polypleksi; lipidipäällysteinen DNA-kompleksi; episomaalinen plasmidi; pieni DNA-rengas; Biofarmaci; Biopharmacy; Biofarmasia; non-viral transfection; retinal pigment epithelium (RPE); lipoplexes; polyplexes; lipid coated DNA complexes (LCDC); episomal plasmid; minicircle; nonviraalinen geeninsiirto; verkkokalvon pigmenttiepiteeli (RPE); lipopleksi; polypleksi; lipidipäällysteinen DNA-kompleksi; episomaalinen plasmidi; pieni DNA-rengas

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Viljamaa, M. (2015). Non-viral gene delivery: Carrier-mediated transfection to retinal pigment epithelial cells and endothelial cells. (Masters Thesis). University of Helsinki. Retrieved from http://hdl.handle.net/10138/153234

Chicago Manual of Style (16th Edition):

Viljamaa, Matleena. “Non-viral gene delivery: Carrier-mediated transfection to retinal pigment epithelial cells and endothelial cells.” 2015. Masters Thesis, University of Helsinki. Accessed December 11, 2019. http://hdl.handle.net/10138/153234.

MLA Handbook (7th Edition):

Viljamaa, Matleena. “Non-viral gene delivery: Carrier-mediated transfection to retinal pigment epithelial cells and endothelial cells.” 2015. Web. 11 Dec 2019.

Vancouver:

Viljamaa M. Non-viral gene delivery: Carrier-mediated transfection to retinal pigment epithelial cells and endothelial cells. [Internet] [Masters thesis]. University of Helsinki; 2015. [cited 2019 Dec 11]. Available from: http://hdl.handle.net/10138/153234.

Council of Science Editors:

Viljamaa M. Non-viral gene delivery: Carrier-mediated transfection to retinal pigment epithelial cells and endothelial cells. [Masters Thesis]. University of Helsinki; 2015. Available from: http://hdl.handle.net/10138/153234

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