Establishment of SV40 Large T-Antigen-Immortalized Yak Rumen Fibroblast Cell Line and the Fibroblast Responses to Lipopolysaccharide

Wang, Junmei; Yue, Ziqi; Che, Li; Li, Hui; Hu, Rui; Shi, Liyuan; Zhang, Xiaohong; Zou, Huawei; Peng, Quanhui; Jiang, Yahui; Wang, Zhisheng

doi:10.3390/toxins15090537

Cited by 1 publication

(2 citation statements)

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“…The HBLV-ZsGreen-Puro vector was purchased from VectorBuilder (Guangzhou, China), and the HBLV-SV40T-3*flag-ZsGreen-Puro lentiviral expression vector was constructed and packaged. The method for establishing immortalized yGC was based on a previous report with minor modifications ( 27 ). Briefly, when the yGC reached 50% confluency, the culture medium was changed to DMEM/F12 with multiple infection numbers (MOIs) of 80 and 5 mg/L of polybrene (Sigma, St. Louis, MO, United States).…”

Section: Methodsmentioning

confidence: 99%

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Establishment of an immortalized yak granulosa cell line: in vitro tool for understanding the molecular processes of ovarian follicle development

Wen,

Zhou,

Yue

et al. 2024

Front. Vet. Sci.

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The yak, a unique species of cattle found exclusively on the western plateau of China, is a valuable source of livelihood for local residents. However, their low fecundity restricts the expansion of yak farming, whereas regional factors limit studies on yak breeding. Granulosa cells (GCs), which provide essential steroid hormones and growth factors for oocytes, have been the focus of many studies on the mechanisms of follicular growth and atresia. This study aimed to establish an immortalized cell line model that could serve as a tool for future studies on the mechanisms of ovarian follicle development in yaks. First, we isolated primary yak granulosa cells (yGCs) and evaluated their replicative senescence after continuous in vitro subculturing. Subsequently, an immortalized culture method for primary yGC was explored, and a new cell line model was established to study the mechanism of follicular development in vitro. We used a mammalian gene expression lentivirus vector to transfer the simian virus 40 large T antigen (SV40T) into primary yGC to obtain an immortalized cell line. The immortalized yGCs were morphologically identical to the primary yGCs, and cell proliferation and growth were normal within a limited number of generations. Follicle-stimulating hormone receptor (FSHR), a specific marker for GCs, was positively expressed in immortalized yGCs. Furthermore, the immortalized yGCs retained the ability of GCs to synthesize estradiol and progesterone and expressed genes related to steroid synthesis. The establishment of immortalized yGC opens up a myriad of possibilities for advancing our understanding of yak reproductive biology and improving yak breeding strategies.

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Section: Methodsmentioning

confidence: 99%

“…Immunofluorescence of ovarian tissue, yGCs, and negative controls (without additional primary antibodies) was performed according to the previous reports, with minor modifications ( 27 , 28 ). Briefly, fresh yak ovaries were fixed in 4% paraformaldehyde for 24 h and embedded in wax, sliced, and dewaxed before antigen retrieval.…”

Section: Methodsmentioning

confidence: 99%