Abstract
Granulosa cells (GCs) are the key components of ovarian follicles and regulate the maturation, communication, growth, and development of oocytes. GCs have great potential as human therapeutic models and in livestock breeding. In this study, we established an immortalized cell line (Im-RGCs) by transforming primary rabbit granulosa cells (Pri-RGCs) with lentivirus-mediated simian virus 40 Large T (SV40LT). Morphologically, Im-RGCs were indistinguishable from Pri-RGCs and maintained intact cell structure as observed by H&E staining. Also, Im-RGCs exhibited no significant change in cell proliferation, viability, and growth. Furthermore, GC-specific markers, such as FSHR, StAR, CYP11A1, and CYP19A1, were examined by PCR, immunofluorescence, and Western blotting. ELISA and karyotype analysis showed that Im-RGCs can synthesize steroid hormones and maintain the normal number of chromosomes during the infinite passage. Furthermore, soft-agar cloning and nude mice tumorigenic experiments indicated the absence of any malignancy transformation in Im-RGCs. In conclusion, we successfully established the immortalized granulosa cell line of rabbit follicles that can be used for biological, animal husbandry, and female reproductive research.
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All data generated or analyzed during this study are included in this published article and its supplementary information files.
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Funding
This study was funded by the Modern Agricultural Industrial System Special Funding (CARS-43-A-1), the earmarked fund for Jiangsu Agricultural Industry Technology System (JATS [2022] 488), and Zhejiang Science and Technology Major Program on Agricultural New Variety Breeding(2021C02068-7).
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Z. B. and Y. C. are responsible for analysis of data and wrote the manuscript. J. L., X. Z., F. W., and Y. D. performed cells experiments. J. C. and Y.R. collected the results, and B. Z. revised the manuscript. Y. C. and X. W. designed the study. All authors checked and confirmed the final manuscript.
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This study was carried out in accordance with the recommendations of Animal Care and Use Committee at Yangzhou University. All experiments were approved by the Animal Care and Use Committee at Yangzhou University (NO.202212006). All methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org) for the reporting of animal experiments.
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Bao, Z., Chen, Y., Li, J. et al. Characterization and establishment of an immortalized rabbit ovary granulosa cell line for reproductive biology experiments. In Vitro Cell.Dev.Biol.-Animal 60, 209–214 (2024). https://doi.org/10.1007/s11626-024-00867-6
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DOI: https://doi.org/10.1007/s11626-024-00867-6