Colorado State University
Salman, Saleh M.
Establishment and characterization of day 30 equine chorionic girdle and allantochorion cell lines.
Degree: MS(M.S.), Animal Sciences, 2019, Colorado State University
Establishing cell lines is a good model for experimental applications to study molecular mechanisms and cell-specific gene expression. A human resistant telomerase reverse transcriptase (hTERT) lentivirus was utilized to establish stable equine embryonic cell lines. Equids have a diffuse epitheliochorial placenta, where the invasive trophoblast is represented by the chorionic girdle (CG) and the noninvasive trophoblast are the allantochorion (AC). Embryonic CG cells are unique to horses compared to other farm animals' embryos. The CG cells are the predecessor of endometrial cups (EC) that differentiate, proliferate, and invade the endometrium by day 38 of pregnancy, yet morphologically, both have similar characteristics supporting the fetal origin for EC. The CG cells have a crucial role in equine chorionic gonadotropin (eCG) production and maintenance of pregnancy during the first trimester. This study has three objectives: 1) establishing a stable cell line from day 30 CG cells and AC using lentivirus encoding hTERT; 2) Characterization of day 30 CG cells and AC cell morphology and expression of eCG alpha (eCGα) and beta (eCGβ) subunits, major histocompatibility complex class II (MHC II), and Kisspeptin receptor (KISS1R) in CG and AC cells; 3) investigating eCG protein production in vitro from day 30 CG and AC cells. Reverse transcriptase (RT) PCR was used to study gene expression in cells and radioimmunoassay (RIA) was used to investigate protein presence in the media. We established a hygromycin-resistant day 30 CG and AC cell lines that express eCGα, eCGβ, and hTERT and confirmed using RT-PCR yielding the predicted bands. The cell lines were maintained for 16 passages, 10 of which were cultured after the lentiviral infection steps. Also, we characterized CG cells as fast-growing, large, binucleated, and epithelioid, and AC cells as rapid-growing showing smaller, squamous, mononucleate, epithelioid, and elongated fibroblastic cells. The RT-PCR results showed eCGα and eCGβ subunits are expressed by both day 30 CG and AC cells, but MHC II and KISS1R genes were not expressed in either of cells. Moreover, RIA results showed that day 30 CG cells did produce eCG protein in vitro earlier than what previous literature has shown. However, day 30 AC cells did not produce eCG protein in vitro, and both CG and AC cell lines stopped secreting eCG in the media after the lentiviral infection.
Advisors/Committee Members: Bruemmer, Jason E. (advisor), Bouma, Gerrit (advisor), Magee, Christianne (committee member), Pinedo, Pablo (committee member), Winger, Quinton (committee member).
Subjects/Keywords: equine; hTERT; trophoblast; girdle; allantochorion; immortalization
to Zotero / EndNote / Reference
APA (6th Edition):
Salman, S. M. (2019). Establishment and characterization of day 30 equine chorionic girdle and allantochorion cell lines. (Masters Thesis). Colorado State University. Retrieved from http://hdl.handle.net/10217/195410
Chicago Manual of Style (16th Edition):
Salman, Saleh M. “Establishment and characterization of day 30 equine chorionic girdle and allantochorion cell lines.” 2019. Masters Thesis, Colorado State University. Accessed August 11, 2020.
MLA Handbook (7th Edition):
Salman, Saleh M. “Establishment and characterization of day 30 equine chorionic girdle and allantochorion cell lines.” 2019. Web. 11 Aug 2020.
Salman SM. Establishment and characterization of day 30 equine chorionic girdle and allantochorion cell lines. [Internet] [Masters thesis]. Colorado State University; 2019. [cited 2020 Aug 11].
Available from: http://hdl.handle.net/10217/195410.
Council of Science Editors:
Salman SM. Establishment and characterization of day 30 equine chorionic girdle and allantochorion cell lines. [Masters Thesis]. Colorado State University; 2019. Available from: http://hdl.handle.net/10217/195410