Establishment and identification of immortalized sheep ovarian granulosa cells

Establishment and identification of immortalized sheep ovarian granulosa cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Establishment and identification of immortalized sheep ovarian granulosa cells Hao Chen, Zhijie Zhao, Xiaona Ding, Pinsheng Li, Bingzhu Zhao, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7555331/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Apr, 2026 Read the published version in BMC Veterinary Research → Version 1 posted 10 You are reading this latest preprint version Abstract Background Follicular granulosa cells are an important cell source for studying animal reproductive function. Results The aim of this study was to investigate the gene transfection of sheep ovarian granulosa cells (GC) mediated by SV40 T lentiviral vector. The effects of GC immortalization and functional properties were verified. The results showed that the cell line could be continuously passaged to the 50th generation. And the cell morphological characteristics and fluorescence expression intensity remained stable. By optimizing the infection conditions (MOI = 10), efficient transfection of GFP-tagged SV40T gene was successfully achieved (efficiency of about 90%)(P<0.05). The immortalized sheep granulosa cell line (GCs-SV40T-GFP) was successfully established. The growth rate of immortalized cells was significantly higher than that of primary GCs-1 cells. Flow cytometry showed that the proportion of S phase and G1 phase of immortalized cells increased significantly (P < 0.05). WB and q-pcr confirmed that the transfected cells continued to secrete estradiol to maintain endocrine function (P < 0.05). In addition, in vivo safety evaluation showed that GCs-SV40T-GFP cells did not induce mouse tumors. Conclusions In this study, a functionally stable immortalized sheep granulosa cell model was successfully constructed. At the same time, it also provides an important tool for animal reproductive function research and hormone regulation mechanism exploration. SV40T lentiviral vector granulosa cells immortalization Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Granulosa cells (GCs) are key functional cells that directly regulate oocyte development and steroid hormone synthesis in mammalian follicles[ 1 ]. In the follicular microenvironment, GCs coordinate the physiological processes of follicular growth, atresia or ovulation by secreting hormones such as estradiol and inhibin[ 2 ]. However, primary GCs generally have problems such as limited proliferation ability and easy aging in vitro culture, and usually lose activity after 5–10 passages, which seriously restricts their application in reproductive biology research[ 3 ], such as dynamic monitoring of hormone secretion, gene function analysis or drug screening model construction[ 4 ]. Therefore, the establishment of a stable and fully functional immortalized GCs model has become an important direction to break through this technical bottleneck[ 5 ]. Immortalization technology endows cells with unlimited proliferation ability by intervening in cell cycle regulation mechanism[ 6 ]. Among them, simian virus 40 large T antigen (SV40T) is widely used in the immortalization of a variety of mammalian cells because of its efficient inhibition of tumor suppressor proteins p53 and Rb[ 7 ]. SV40T drives cells to enter the S phase by removing cell cycle checkpoints, thereby bypassing replicative senescence[ 8 ]. This technology has been successfully applied to a variety of cell lines in human, mouse and bovine, but the research in sheep GCs is still extremely limited[ 9 ]. In addition, the immortalization process may be accompanied by abnormal cell function (such as decreased hormone secretion) or potential tumorigenic risk, which needs systematic verification[ 10 ]. Lentiviral vector has become the preferred tool for gene delivery due to its high transfection efficiency, stable genome integration and low immunogenicity[ 11 ]. In this study, the lentiviral vector of SV40T and green fluorescent protein (GFP) fusion was constructed, and the multiplicity of infection (MOI = 10) was optimized to achieve efficient transfection of sheep GCs (efficiency of 90%), and an immortalized cell line (GCs-SV40T-GFP) was established[ 12 ]. Preliminary experiments confirmed that the transfected cells remained stable in morphology and fluorescence expression after continuous passage to the 50th generation, which preliminarily verified the long-term integration and expression stability of SV40T gene[ 13 ]. Although SV40T immortalization technology has significant advantages, its application still faces two major challenges: one is the maintenance of cell function after immortalization[ 14 ]. For example, immortalized human mesenchymal stem cells can retain the ability to differentiate, while some hepatocytes have reduced metabolic activity. For GCs, whether the synthesis ability of its core function-steroid hormones (such as estradiol) is impaired directly affects the research value of immortalized cells. The second is the potential tumorigenicity of SV40T[ 15 ][ 16 ]. Therefore, this study not only needs to verify the hormone secretion function of GCs-SV40T-GFP, but also needs to evaluate its biosafety through in vivo transplantation experiments[ 17 ]. As an important economic animal and biomedical model, the reproductive physiology of sheep is of great significance to the development of animal husbandry breeding and human assisted reproductive technology. However, the short life characteristics of sheep GCs limit the depth of in vitro research[ 18 ]. In this study, an immortalized GCs-SV40T-GFP cell line was constructed to provide a stable and functional experimental model for analyzing the potential effects of follicular development mechanism, hormone regulation network and environmental pollutants on reproductive function in sheep[ 19 ]. In addition, if the model is proved to be safe and reliable, it can provide technical support for improving animal reproduction efficiency[ 20 ]. In summary, this study focused on SV40T lentiviral vector-mediated sheep granulosa cell immortalization technology, and systematically evaluated its proliferation characteristics, functional stability and biosafety, aiming to fill the technical gaps in this field and provide innovative tools for reproductive biology research and clinical application[ 21 ]. 2. Materials and methods 2.1. Treatment of GCs In this study, 10 ovaries of 2-year-old healthy ewes were collected from the slaughterhouse in Tianzhu County, Gansu Province, and stored in 37° normal saline with penicillin and streptomycin (0.48g) added to the laboratory. Follicular fluid was extracted from ovarian follicles with a diameter of about 3 ~ 8 mm and injected into the tube. The filtrate was filtered with a 100 nm filter membrane, and then the follicular fluid was filtered with a 70 nm filter membrane, and then centrifuged with a high-speed centrifuge (TG16-WS desktop high-speed centrifuge) at 1500r/min to collect the precipitate. The cells were resuspended in DMEM medium containing 10% fetal bovine serum. Cell morphology was observed and cultured in a cell incubator at 37°C and 5% CO2. 2.2. Cell counting plate method In this study, sheep granulosa cells were cultured in vitro, and the cell proliferation dynamics within 96 hours were systematically monitored by cell counting plate method. The specific method was as follows : synchronized granulosa cells were cultured in DMEM/F12 complete medium, and samples were taken every 12 hours from 0 h. Trypsin digestion and background trypan blue staining were used to count living cells under an inverted microscope using a modified Neubauer counting plate. Biological and technical repetitions were set at each time point. Finally, the cell density was calculated and the growth curve was drawn, and the population doubling time was calculated. 2.3. Immunofluorescence staining The primary cultured GCs were seeded in 24-well plates, and the culture was terminated when the cells reached 80% confluence. Then immunofluorescence detection was performed. GCs were fixed with 4% paraformaldehyde for 30 min, and 0.1% Triton X-100 permeabilized the cell membrane for 15 min. It was blocked with 5% BSA (Gibco, Carlsbad, CA) at room temperature for 2 h. Cells were incubated with rabbit monoclonal antibody Vimentin at a concentration of 1:200 (Bioss, Beijing, China) overnight at 4°C. The cells were incubated with anti-rabbit secondary antibody at a concentration of 1:500 (Bioss, Beijing) and incubated at room temperature for 1 h the next day. Blank control was only incubated with secondary antibody. DAPI was used for nuclear re-staining. Cells were briefly washed three times with PBS between the start, end, and each step of staining. Immunostaining was evaluated by fluorescence microscopy. This experiment was repeated three times. 2.4. Confirmation of GFP-GCs (multiplicity of infection) in sheep granulosa cells On the first day, SV40T sheep ovarian granulosa cells were inoculated in a 12-well plate containing 1E5 cells. The next day, the GFP virus stock solution ( Cegrogen Shanghai ) was taken out from the − 80°C refrigerator and melted in an ice bath before infection. The virus GFP stock solution was diluted with 800µL complete medium with MOI = 1,10,50,100, and the original medium of the treatment group was removed. 500µL medium containing lentivirus dilution was added to the cells in the treatment group. The culture medium was changed on the third day, and the culture medium containing lentivirus was changed to 1 m L complete culture medium after 16 h of infection. On the 5th day, the infection efficiency was detected. The fluorescence was observed under an inverted fluorescence microscope, and the effect of lentivirus infection on target cells was calculated by fluorescence intensity. 2.5. SV40T-GFP-GCs lentivirus transfection On the first day, SV40T sheep ovarian granulosa cells were inoculated in 12-well plates with 1E5 cells. The next day, before infection, the virus stock solution was taken out from the-80°C refrigerator and melted in an ice bath. The virus stock solution was diluted with 80µL complete medium at MOI = 10, and the original medium of the treatment group was removed. The 500µL medium containing the lentivirus dilution was added to the cells of the treatment group. On the third day, the culture medium was replaced, and the culture medium containing lentivirus was replaced with 1 mL complete culture medium after 16 h of infection. On the 5th day, the infection efficiency was detected, and the fluorescence was observed under an inverted fluorescence microscope to determine the effect of lentivirus infection on target cells. The appropriate eukaryotic resistance screening cells were selected (the complete lethal concentration of puromycin was 3µg/mL, and the maintenance concentration was 1.5µg/mL). The cells were screened for two rounds of common drugs ( one round for 2 days, determined by antibiotic pre-experiment ). The cells could be stably passaged. After stable passage, DMEM + 10% FBS + 1%PS + 1.5µg/mL puromycin complete medium was used to maintain the cells. 2.6. RNA isolation, reverse transcription and quantitative RT-PCR. Total RNA was extracted using the Trizol method (SOLEIBO, Beijing, China), and the concentration and purity of RNA samples were evaluated using an ultra-micro UV-visible spectrophotometer (NanoReady/FC-1100) spectrophotometer. RNA samples with D260/D280 values between 1.8 and 2.0 were selected for cDNA synthesis using a PrimeTM TMRT kit (Takara, Beijing, China) with 1 µg of high-purity RNA. SYBR PreMix Ex Taq TM II (Takara, Beijing, China) and real-time PCR system (Bio-Rad, Hercules, CA, USA) were used for qRT-PCR. The reaction conditions are as follows: pre-denaturation at 95°C for 3 minutes, followed by denaturation at 95°C for 10 seconds, and annealing at 60°C for 30 seconds, for a total of 40 cycles. Each group consists of three biological replicates. The relative expression was calculated by 2-ΔΔCt. According to the gene sequence of sheep in GenBank database, primers were designed by Primer Premier 5.0 software for real-time fluorescence quantitative PCR. The primers were synthesized by Beijing Huada Gene Information Company (Table 1 ). Table 1 GCs-SV40T Real-time PCR primer sequences Gene name Sequences (5’→3’) Length(bp) Accession number CYP19A1 F-ATGCTGGTGCTGAGTATGTGGT 192 NM_001123000.1 R-GCTGACAATCTTGAGGGTGTTG ACTIN F-ATATTGCTGCGCTCGTGGTT 224 NM_001009784.3 R-GTTGGTGACAATGCCGTGCT SV40T F-TGGAAACCAAGTGCGACGAC R-CGGCGAAGATAGCGGCATTA ACTB-F F-ATTGCTGACAGGATGCAGAAGG R-GCTGGAAGGTGGACAGTGAGG 2.7. Flow cytometry GCs-1, SV40T-GFP-GCs-1, SV40T-GFP-GCs-10, SV40T-GFP-GCs-25, SV40T-GFP-GCs-50 were added to 200µL cell suspension and added to 6-well plates. Add 10% fetal bovine serum complete medium, pre-incubated at 37°C, 5% CO2 incubator for 48 hours. Rise to about 90 percent, trypsin digestion and centrifugation. The stained cell suspension was transferred to a flow tube, and the data were detected and analyzed by flow cytometry. 2.8. ELISA detection In order to reduce the influence of fetal bovine serum, phenol red-free medium was used to avoid estrogen interference, and activated carbon/dextran-treated double-antibody FBS was replaced. Before the formal experiment, 24-hour serum starvation was performed and replaced with 0.5-1% stripped FBS medium. 28.6ng/m LA4 (androstenedione) was added. The primary, 10th, 25th and 50th generations were recorded respectively. When they grew to 100% of the cells, the complete medium was collected in a 1.5mL centrifuge tube. After centrifugation at 3000 rpm for 15minutes, the supernatant was taken. Concentrations were determined using kits (Shanghai, China, Mlbio). 2.9. West-blot assay The treated cells were collected and lysate and PMSF (100:1, Servicebio, China) were added to the cell precipitate. Lysis was performed on ice for 30 min followed by centrifugation at 10,000×g at 4℃for 15min. Protein concentration was determined using the BSA kit (Servicebio, China). The protein loading volume was 50mg. Separation was performed by electrophoresis using 10% SDS-PAGE followed by electrotransfer to polyvinylidene difluoride membranes (MCE, New Jersey, USA). The membranes were closed in 5% skim milk in TBS/Tween for 1 h at room temperature and then mixed with FHSR(Servicebio, China) 1:2000 and β-actin (Servicebio, China) 1:3000 with skim milk-PBST (Servicebio, China) and co-incubated. The membranes were then incubated with HRP conjugated antirabbit or anti-mouse secondary antibody 1:1000 (Servicebio, China) with 5% skim milk-PBST and detected by the Western Lighting ECL detection system. Signals were quantified using Image J 2020. 2.10. Tumorigenicity experiment in mice Nine 5-week-old male BALB/c nude mice were purchased from Lanzhou Veterinary Research Institute and randomly divided into 3 groups with 3 mice in each group. After 1 week of adaptive culture, primary granulosa cells, 50th generation immortalized granulosa cells and HeLa cells were inoculated respectively. The cells were placed in two T25 culture flasks and cultured in DMEM medium containing 20% fetal bovine serum. The incubator was set at 37°C, 5% carbon dioxide. Until the cell growth is greater than 98%. After trypsin digestion, each generation of cells was adjusted to 1 × 7 Ωcells / mL, and 10 µL was injected subcutaneously into the left back of mice. After 4 weeks of continuous observation, the tumor size was monitored with a ruler. 2.11. Statistical analysis All data represent at least three independent experiments (with comparable results). ANOVA was used to evaluate the statistical significance of the data. The difference of P < 0.05 was considered statistically significant. One-way analysis of variance was used to analyze the data using GraphPad Prism 8 software, and paired comparisons were performed using LSD and Tukey tests. The results are expressed as mean ± standard deviation. 3. Results Successful isolation and identification of GCs cells. The ovaries of healthy sheep were collected, stored at 37°C, and returned to the laboratory within 4 h. Follicular fluid was collected by puncturing follicles, and then follicular fluid was filtered with a 100u + 70u filter. Primary culture was performed, and the first generation of cells was used for the experiment (Figure.1A). The cell growth density at different times was measured by cell counting (Figure.1B). The FSHR protein receptor was stained by immunofluorescence staining to verify that the cells were the required granulosa cells (Figure.1C). Confirmation of fluorescent GFP-GCs. The GFP virus was used to determine the MOI, and the light expression efficiency of sheep ovarian granulosa cells reached 90% at MOI = 10, and the pre-experiment passed. Subsequent experiments will be infected with MOI = 10. When the second generation of cells grew to 80%, transfection was performed (P<0.05)(Fig. 2A,B). SV40T-GFP-GCs lentivirus was successfully transfected and the morphology was stable. Cell transfection was performed using SV40T-GFP virus with MOI = 10. When the cells grew to 80%, SV40T-GFP transfection was performed (Fig. 3A). The transfection effect of SV40T-GFP-GCs was detected by q-PCR and fluorescence expression (P < 0.05) (Fig. 3B). Cell growth was monitored by cell counting plate method (Figure.3C). The morphology of SV40T-GFP-GCs (primary, 1st, 10th, 25th and 50th generations) did not change, and the fluorescence was stably expressed, indicating that the SV40T-GFP gene could be stably expressed (Fig. 3D). The cell viability of SV40T-GFP-GCs was increased. Cell cycle was determined by flow cytometry. After the first generation, the S and G1 phase of the 10th generation of SV40T-GFP-GCs cells increased significantly. The S and G1 phases of the 25th and 50th generation of cells were also the same as those of GC (P<0.05)(Fig. 4A-C). The ability of SV40T-GFP-GCs cells to produce estradiol was enhanced. The production of estradiol in SV40T-GFP-GCs cells after transfection was detected by ELISA (P < 0.05) (Fig. 5A). Compared with the first generation of GCs-1, the estradiol content of SV40T-GFP-GCs-1 and SV40T-GFP-GCs-10 cells increased significantly, and the 25th and 50th generations were comparable to GCs-1 (P < 0.05) (Fig. 5B-C). The expression of CYP19a1 in SV40T-GFP-GCs was significantly enhanced by WB and q-pcr. Detection of SV40T-GFP-GCs cells do not have tumorigenicity. GCs-1, SV40T-GFP-GCs-50, HELA cells were set as control (Fig. 6A,B). After 4 weeks of culture, the Hela cells had obvious tumors. GCs-1 cells and SV40T-GFP-GCs-50 cells had no tumors (Fig. 5C-E). Flow cytometry was used to detect the blood of mice, MDSC, PDL1, CD68 cells increased significantly (P<0.05)(Fig. 6F-H). 4. Discussion In this study, an immortalized sheep granulosa cell line (GCs-SV40T-GFP) was successfully constructed by SV40T lentiviral vector, and its proliferation characteristics, functional stability and biosafety were systematically evaluated[ 22 ]. The experimental results are discussed from four aspects: technical advantages, function maintenance mechanism, security disputes and future application prospects[ 23 ]. In this study, the transfection efficiency of SV40T gene mediated by lentiviral vector was as high as 90% (MOI = 10), and the transfected cells still maintained stable morphology and fluorescence expression after continuous passage to the 50th generation[ 24 ]. This result highlights the significant advantages of lentiviral vectors in gene delivery: their genomic integration ensures long-term expression of foreign genes, while low immunogenicity reduces interference with cell activity. Compared with traditional plasmid transfection or retrovirus, the ability of lentivirus to infect non-dividing cells further improves its application potential in primary cell immortalization. In addition, the introduction of GFP tags provides an intuitive tool for real-time monitoring of transfection efficiency and gene stability during cell passage. This design is innovative in similar studies[ 25 ]. It is worth noting that SV40T significantly enhances the proliferation of GCs by targeting p53 and Rb proteins to relieve cell cycle arrest[ 26 ]. Cell cycle analysis showed that the proportion of S phase cells in GCs-SV40T-GFP increased, which was consistent with the results of human immortalized mesenchymal stem cells. However, the response of sheep GCs to SV40T may be species-specific[ 27 ]. For example, the increase in cell proliferation rate in this study was higher than that in some bovine or mouse cell models, which may be related to the low original proliferation potential of sheep GCs. This finding suggests that the effect of SV40T immortalization is not only dependent on gene delivery efficiency, but also closely related to the biological characteristics of target cells[ 28 ]. Although immortalization is often accompanied by abnormal cell function, this study found that GCs-SV40T-GFP can continue to secrete estradiol, and the secretion level is not significantly different from that of primary cells. This result is consistent with the study of immortalized human luteal granulosa cells, indicating that SV40T may not interfere with steroidogenesis-related pathways (such as CYP19A1 expression or StAR protein activity)[ 29 ]. It is further speculated that although the inhibition of p53 by SV40T may affect the DNA repair mechanism, it does not significantly destroy the core regulatory network of endocrine function of GCs. However, the potential impact of long-term passage on functional stability needs to be vigilant. For example, some immortalized hepatocytes have decreased metabolic enzyme activity after passage, which may be related to the accumulation of epigenetic modifications or mitochondrial dysfunction. Therefore, in the future, it is necessary to deeply analyze the global effect of SV40T on GCs signaling pathway in sheep through transcriptome or metabolomics analysis[ 30 ]. In addition, the secretion dynamics of other hormones (such as progesterone or inhibin) were not detected in this study. Sheep GCs have functional heterogeneity at different developmental stages of follicles, and whether immortalization causes cells to ' lock ' into specific functional states remains to be verified. If GCs-SV40T-GFP can still respond to gonadotropin (such as FSH) stimulation and regulate hormone synthesis, the model can be used to simulate the dynamic process of follicular development and has higher research value[ 31 ]. The risk of tumorigenicity of SV40T is the core issue that limits its clinical application. In this study, we confirmed that GCs-SV40T-GFP did not induce tumor formation by in vivo transplantation experiments in mice. This result contradicts some studies: for example, SV40T immortalized human fibroblasts can form sarcomas in immunodeficient mice[ 32 ]. The difference may be due to the following reasons: (1) Species specificity: the tumorigenic transformation threshold of sheep GCs may be higher than that of human cells; (2) Gene expression regulation: The promoter activity or copy number control of SV40T in lentiviral vector may reduce its carcinogenic potential. (3) Cell type difference: GCs themselves are terminally differentiated cells, and their malignant transformation requires additional driver mutations. Nevertheless, long-term safety still needs to be carefully evaluated. It is recommended that follow-up studies extend the observation period and use more sensitive detection methods (such as circulating tumor DNA analysis) to exclude the possibility of small lesions[ 33 ]. It is worth noting that combined immortalization strategies (such as co-expression of SV40T and hTERT) have been shown to reduce genomic instability. If hTERT is introduced into sheep GCs, it may further optimize the genetic stability of the cell line and reduce the dose-dependent toxicity of SV40T[ 34 ]. The GCs-SV40T-GFP cell line constructed in this study provides an important tool for the study of sheep reproductive biology. For example, this model can be used to:(1) analyze the function of key genes for follicular development (such as FOXL2 or AMH) ; (2) Screening natural compounds or drugs that regulate the synthesis of estradiol ; (3) Assess the toxic effects of environmental pollutants (such as bisphenol A) on ovarian function. In addition, in the field of animal husbandry, this cell line can be used to optimize oocyte maturation conditions in in vitro fertilization (IVF) technology, or to develop a high-throughput screening platform for ovulation-promoting drugs. Compared with cattle or mouse models, data on GCs immortalization in sheep are still scarce. The success of this study provides a technical reference for the establishment of other large livestock germ cell models. For example, combined with CRISPR-Cas9 gene editing technology, specific gene knockout or mutation models can be constructed in immortalized cell lines to study the molecular mechanism of reproductive disorders. 5. Conclusions This study confirmed that SV40T lentiviral vector can efficiently realize the immortalization of sheep granulosa cells, and the immortalized cells meet the basic research needs in terms of proliferation ability, functional characteristics and biosafety. However, the following issues still need to be further explored:(1) The effect of immortalization on the epigenetic characteristics and stress response ability of GCs ;(2) The functional recovery efficiency of cell lines after long-term cryopreservation and resuscitation;(3) Synergistic effect of SV40T with other immortalized genes (such as hTERT). Future work can combine multi-omics technology with in vitro and in vivo functional verification to comprehensively evaluate the reliability of the cell line and expand its practical application in reproductive medicine and animal husbandry. In conclusion, the establishment of GCs-SV40T-GFP cell line not only fills the technical gap in the in vitro research model of sheep germ cells, but also lays a solid foundation for further exploring the regulation mechanism of follicular development and developing new reproductive intervention strategies. Declarations Conflict of interests The authors declare that they have no conflicts of interest regarding this work. Animal ethics statement All experimental animal operations were performed in full accordance with institutional guidelines and approved by the Ethics Committee of the School of Life Sciences and Engineering of Northwest Minzu University (xbmu-sm-202593). Funding This research was supported by the National Natural Science Foundation of China [32460907], the Plateau Animal Disease Innovation Team [20240036], the Lanzhou Science and Technology Plan Project [No. 2022-2-­44] Colour artwork Colour should be used only in online version. Data statement The data that support the findings of this study are available from the corresponding author upon reasonable request. CRediT authorship contribution statement Chen Hao : designed the study, performed the experiments, and, Writing-original draft, the manuscript. Zhao Zhijie : performed the experiments and, Writing-original draft, the manuscript. Ding Xiaona : performed the experiments and, Writing-original draft, the manuscript. Li Pinsheng: designed the study, performed the experiments, and, Writing-original draft, the manuscript. Zhao Bingzhu : per-formed the experiments and, Writing-original draft, the manu-script. Yuan Yue : performed the experiments and, Writing-original draft, the manuscript. Rui Xiao : designed the study, performed the experiments, and, Writing-original draft, the manuscript. Zhang Taojie : hel-ped in performing the experiments and, Formal analysis, the, Data curation. Yingpai Zhaxi : designed the study and helped in, Writing-original draft, the manuscript. Shengdong Huo : designed the study and helped in, Writing-original draft, the manuscript. Declaration of competing nterest Declarations of interest: none. Acknowledgements Thank Professor Huo Shengdong for his help. References Hayakawa T. Establishment and characterization of immortalized sweat gland myoepithelial cells. Sci Rep 2022. Kwack MH, Hamida OB, Kim MK, Kim MK, Sung YK. Establishment and characterization of matched immortalized human frontal and occipital scalp dermal papilla cell lines from androgenetic alopecia. Sci Rep 2023;13:21421. https://doi.org/10.1038/s41598-023-48942-4. Ma T, Liu W, Jiang D, Zhang G, Zhao X, Zhang Y, et al. Analysis of Toxic Effects of Fluoride on Ovine Follicular Granulosa Cells Using RNA-Seq. Antioxidants 2024;13:506. https://doi.org/10.3390/antiox13050506. Zhang L, Liu K, Liu Z, Tao H, Fu X, Hou J, et al. In pre-clinical study fetal hypoxia caused autophagy and mitochondrial impairment in ovary granulosa cells mitigated by melatonin supplement. J Adv Res 2024;64:15–30. https://doi.org/10.1016/j.jare.2023.11.008. Lin S. Establishment methods and research progress of livestock and poultry immortalized cell lines: A review. Front Vet Sci n.d. Matsumura T, Takesue M, Westerman KA, Okitsu T, Sakaguchi M, Fukazawa T, et al. ESTABLISHMENT OF AN IMMORTALIZED HUMAN-LIVER ENDOTHELIAL CELL LINE WITH SV40T AND hTERT: Transplantation 2004;77:1357–65. https://doi.org/10.1097/01.TP.0000124286.82961.7E. Tan J-J, Wang L, Mo T-T, Dai Y-F, Lu J, Liu X, et al. Establishment of Immortalized Laryngeal Epithelial Cells Transfected with Bmi1. Cell Transplant n.d. Wang J, Hu R, Wang Z, Guo Y, Wang S, Zou H, et al. Establishment of Immortalized Yak Ruminal Epithelial Cell Lines by Lentivirus-Mediated SV40T and hTERT Gene Transduction. Oxid Med Cell Longev 2022;2022:1–17. https://doi.org/10.1155/2022/8128028. Zieliński M, Romanik-Chruścielewska A, Mikiewicz D, Łukasiewicz N, Sokołowska I, Antosik J, et al. Expression and purification of recombinant human insulin from E. coli 20 strain. Protein Expr Purif 2019;157:63–9. https://doi.org/10.1016/j.pep.2019.02.002. Guo L, Wang Z, Li J, Li J, Cui L, Dong J, et al. Immortalization effect of SV40T lentiviral vectors on canine corneal epithelial cells. BMC Vet Res 2022;18:181. https://doi.org/10.1186/s12917-022-03288-3. Seridi N, Hamidouche M, Belmessabih N, El Kennani S, Gagnon J, Martinez G, et al. Immortalization of primary sheep embryo kidney cells. Vitro Cell Dev Biol - Anim 2021;57:76–85. https://doi.org/10.1007/s11626-020-00520-y. Norris N, Yau B, Kebede MA. Isolation and Proteomics of the Insulin Secretory Granule. Metabolites 2021;11:288. https://doi.org/10.3390/metabo11050288. Li R-F, Nan G-X, Wang D, Gao C, Yang J, He T-C, et al. SV40T reprograms Schwann cells into stem-like cells that can re-differentiate into terminal nerve cells. Int J Dev Biol 2021;65:557–62. https://doi.org/10.1387/ijdb.210062zz. Yang C, Lan W, Ye S, Zhu B, Fu Z. Transcriptomic Analyses Reveal the Protective Immune Regulation of Conjugated Linoleic Acids in Sheep Ruminal Epithelial Cells. Front Physiol 2020;11:588082. https://doi.org/10.3389/fphys.2020.588082. Lenz LS, Wink MR. The other side of the coin: mesenchymal stromal cell immortalization beyond evasion of senescence. Hum Cell 2023;36:1593–603. https://doi.org/10.1007/s13577-023-00925-3. He H, Su X, Yang H, Zhang Y, Duan C, Yang R, et al. Effects of prolactin on the proliferation and hormone secretion of ovine granulosa cells in vitro. Anim Biosci 2024;37:1712–25. https://doi.org/10.5713/ab.23.0448. Oshimura M, Tabata T, Uno N, Takata S, Hichiwa G, Kanazawa I, et al. Rejuvenation of human mesenchymal stem cells using a nonintegrative and conditionally removable Sendai virus vector. Sci Rep 2024;14:23623. https://doi.org/10.1038/s41598-024-74757-y. Zhan K, Yang TY, Chen Y, Jiang MC, Zhao GQ. Propionate enhances the expression of key genes involved in the gluconeogenic pathway in bovine intestinal epithelial cells. J Dairy Sci 2020;103:5514–24. https://doi.org/10.3168/jds.2019-17309. Bruscoli S, Febo M, Riccardi C, Migliorati G. Glucocorticoid Therapy in Inflammatory Bowel Disease: Mechanisms and Clinical Practice. Front Immunol 2021;12:691480. https://doi.org/10.3389/fimmu.2021.691480. Primak A, Kalinina N, Skryabina M, Usachev V, Chechekhin V, Vigovskiy M, et al. Novel Immortalized Human Multipotent Mesenchymal Stromal Cell Line for Studying Hormonal Signaling. Int J Mol Sci 2024;25:2421. https://doi.org/10.3390/ijms25042421. Dos Santos A, Lyu N, Balayan A, Knight R, Zhuo KS, Sun Y, et al. Generation of Functional Immortalized Human Corneal Stromal Stem Cells. Int J Mol Sci 2022;23:13399. https://doi.org/10.3390/ijms232113399. Albericio G, Higuera M, Araque P, Sánchez C, Herrero D, García-Brenes MA, et al. Development of a Bmi1+ Cardiac Mouse Progenitor Immortalized Model to Unravel the Relationship with Its Protective Vascular Endothelial Niche. Int J Mol Sci 2024;25:8815. https://doi.org/10.3390/ijms25168815. Chen H, Li J, Pan X, Hu Z, Cai J, Xia Z, et al. A novel avian intestinal epithelial cell line: its characterization and exploration as an in vitro infection culture model for Eimeria species. Parasit Vectors 2024;17:25. https://doi.org/10.1186/s13071-023-06090-8. Li J, Zhao B, Zhang X, Dai Y, Yang N, Bao Z, et al. Establishment and functional characterization of immortalized rabbit dermal papilla cell lines. Anim Biotechnol 2023:1–10. https://doi.org/10.1080/10495398.2023.2252861. Chen H, Wang J, Zhao B, Yang Y, Yang C, Zhao Z, et al. N-Acetylcysteine relieving hydrogen peroxide-induced damage in granulosa cells of sheep. Cell Adhes Migr 2025;19:2484182. https://doi.org/10.1080/19336918.2025.2484182. Zhao S, Cui H, Fang X, Xia W, Tao C, Li J. Increased DNMT1 acetylation leads to global DNA methylation suppression in follicular granulosa cells during reproductive aging in mammals. BMC Genomics 2024;25:1030. https://doi.org/10.1186/s12864-024-10957-0. Habara O, Logan CY, Kanai-Azuma M, Nusse R, Takase HM. WNT signaling in pre-granulosa cells is required for ovarian folliculogenesis and female fertility. Development 2021;148:dev198846. https://doi.org/10.1242/dev.198846. Tian H, Ren P, Liu K, Qiu C, Fan L, Li J, et al. Transcriptomic comparison of ovarian granulosa cells between adult sheep and prepubertal lambs. BMC Genomics 2022;23:151. https://doi.org/10.1186/s12864-022-08379-x. Masumi S, Lee EB, Dilower I, Upadhyaya S, Chakravarthi VP, Fields PE, et al. The role of Kisspeptin signaling in Oocyte maturation. Front Endocrinol 2022;13:917464. https://doi.org/10.3389/fendo.2022.917464. Fang X, Xia W, Li S, Qi Y, Liu M, Yu Y, et al. SIRT2 Is Critical for Sheep Oocyte Maturation through Regulating Function of Surrounding Granulosa Cells. Int J Mol Sci 2022;23:5013. https://doi.org/10.3390/ijms23095013. Yang R, Duan C, Zhang S, Liu Y, Zhang Y. Prolactin Regulates Ovine Ovarian Granulosa Cell Apoptosis by Affecting the Expression of MAPK12 Gene. Int J Mol Sci 2023;24:10269. https://doi.org/10.3390/ijms241210269. Abdurahman A, Aierken W, Zhang F, Obulkasim R, Aniwashi J, Sulayman A. miR-1306 induces cell apoptosis by targeting BMPR1B gene in the ovine granulosa cells. Front Genet 2022;13:989912. https://doi.org/10.3389/fgene.2022.989912. Zhai B, Li X, Zhao Z, Cao Y, Liu X, Liu Z, et al. Melatonin Protects the Apoptosis of Sheep Granulosa Cells by Suppressing Oxidative Stress via MAP3K8 and FOS Pathway. Genes 2023;14:1067. https://doi.org/10.3390/genes14051067. Dai T, Kang X, Yang C, Mei S, Wei S, Guo X, et al. Integrative Analysis of miRNA-mRNA in Ovarian Granulosa Cells Treated with Kisspeptin in Tan Sheep. Animals 2022;12:2989. https://doi.org/10.3390/ani12212989. Additional Declarations No competing interests reported. Supplementary Files GraphicalAbstract.jpg Cite Share Download PDF Status: Published Journal Publication published 21 Apr, 2026 Read the published version in BMC Veterinary Research → Version 1 posted Editorial decision: Revision requested 23 Oct, 2025 Reviews received at journal 21 Oct, 2025 Reviewers agreed at journal 10 Oct, 2025 Reviews received at journal 05 Oct, 2025 Reviewers agreed at journal 25 Sep, 2025 Reviewers invited by journal 16 Sep, 2025 Editor assigned by journal 16 Sep, 2025 Editor invited by journal 15 Sep, 2025 Submission checks completed at journal 12 Sep, 2025 First submitted to journal 12 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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13:20:16","extension":"html","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":107161,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7555331/v1/2eecb885ed466e0b77397d85.html"},{"id":92180245,"identity":"a70a6f02-b4ea-4022-ac0f-0f703cc5d12d","added_by":"auto","created_at":"2025-09-25 13:28:15","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":552427,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCulture and identification of the first generation of GCs cells:\u003c/strong\u003e (A) Cell growth and identification. (B) Cell growth time and density (C-E) Immunofluorescence detection of fshr in GCs cells.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7555331/v1/448deef242ac6bdec989c6c6.jpg"},{"id":92178842,"identity":"6dc25ce3-1b22-493c-85f9-d8285ef519dc","added_by":"auto","created_at":"2025-09-25 13:20:15","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":798664,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQuantification of multiplicity of infection (MOI): \u003c/strong\u003e(A-B) MOI was set at 1,10,50,100 different concentrations. When MOI=10, the transfection efficiency reached 90%, which could be used in the experiment(P＜0.05).\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7555331/v1/3f526936c2103bdc9cfe3f45.jpg"},{"id":92178846,"identity":"60012e59-654c-4cdc-a92d-11519ee8dfbb","added_by":"auto","created_at":"2025-09-25 13:20:15","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":259424,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSV40T-GFP lentivirus transfection:\u003c/strong\u003e (A) The first generation of 96 h GCs cells were transfected into the first generation of SV40T-GFP-GCs-1 cells, and the fluorescence expression was obvious. (B) qpcr was used to detect the high expression of SV40T-GFP-GCs-1 relative to GCs-1 gene. (C) During the subsequent passage, the cell morphology did not change. SV40T-GFP gene still expressed high fluorescence. (D) The cell growth time after transfection was significantly shortened(P＜0.05).\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7555331/v1/39d7bff4a69c2dfab8cbeede.jpg"},{"id":92178845,"identity":"593bb313-8dc7-4c92-aae7-d12e1274b421","added_by":"auto","created_at":"2025-09-25 13:20:15","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":583973,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetection of proliferation activity of SV40T-GFP-GCs after transfection:\u003c/strong\u003e (A-C) Compared with GCs-1, the S+G1 phase of SV40T-GFP-GCs after transfection of the first and tenth generations increased significantly, and the proliferation activity increased significantly. The proliferation activity of GCs-1, S+G1 phase cells was slightly increased after transfection of the 25th and 50th generations(P＜0.05).\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7555331/v1/d14bbf52dda7bb1dfbb10b9c.jpg"},{"id":92178847,"identity":"0f3e5d5d-d251-443b-b455-9a7a70133cb9","added_by":"auto","created_at":"2025-09-25 13:20:15","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":174062,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetection of estradiol production in SV40T-GFP-GCs cells after transfection: \u003c/strong\u003e(A) The estradiol of SV40T-GFP-GCs-10, SV40T-GFP-GCs-25 was significantly higher than that of GCs-1 and the SV40T-GFP-GCs-50 band was flat. (B-C) The expression level of CYP19a1 protein was detected by WB and q-pcr. There were significant differences in the groups represented by different letters (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7555331/v1/03aba9d93c6afe80e748ede3.jpg"},{"id":92180246,"identity":"d8c13866-b5db-4fed-9996-df7f2b7b4bff","added_by":"auto","created_at":"2025-09-25 13:28:15","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2568626,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTransfected SV40T-GFP-GCs mice do not have tumorigenicity:\u003c/strong\u003e (A) cell morphology (B) growth density. (C-E) Mice inoculated with HELA cells had obvious tumors, GCs-1, SV40T-GFP-GCs-50, and did not have tumors. (F-H) MDSC, PDL1, CD68 cells increased significantly(P＜0.05).\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7555331/v1/5102c1dc98813da82d21fc48.jpg"},{"id":107927743,"identity":"cf6cba74-50f1-49f0-b504-98ce9dc619f9","added_by":"auto","created_at":"2026-04-27 16:03:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5183670,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7555331/v1/43859f92-d085-4d2e-9b9d-12ee58bb5873.pdf"},{"id":92178844,"identity":"61aacde4-c094-456a-9722-936e6b6c0aeb","added_by":"auto","created_at":"2025-09-25 13:20:15","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":98328,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7555331/v1/384712dc06c4c2ec5f286159.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Establishment and identification of immortalized sheep ovarian granulosa cells","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eGranulosa cells (GCs) are key functional cells that directly regulate oocyte development and steroid hormone synthesis in mammalian follicles[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In the follicular microenvironment, GCs coordinate the physiological processes of follicular growth, atresia or ovulation by secreting hormones such as estradiol and inhibin[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, primary GCs generally have problems such as limited proliferation ability and easy aging in vitro culture, and usually lose activity after 5\u0026ndash;10 passages, which seriously restricts their application in reproductive biology research[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], such as dynamic monitoring of hormone secretion, gene function analysis or drug screening model construction[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Therefore, the establishment of a stable and fully functional immortalized GCs model has become an important direction to break through this technical bottleneck[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eImmortalization technology endows cells with unlimited proliferation ability by intervening in cell cycle regulation mechanism[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Among them, simian virus 40 large T antigen (SV40T) is widely used in the immortalization of a variety of mammalian cells because of its efficient inhibition of tumor suppressor proteins p53 and Rb[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. SV40T drives cells to enter the S phase by removing cell cycle checkpoints, thereby bypassing replicative senescence[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This technology has been successfully applied to a variety of cell lines in human, mouse and bovine, but the research in sheep GCs is still extremely limited[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In addition, the immortalization process may be accompanied by abnormal cell function (such as decreased hormone secretion) or potential tumorigenic risk, which needs systematic verification[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eLentiviral vector has become the preferred tool for gene delivery due to its high transfection efficiency, stable genome integration and low immunogenicity[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In this study, the lentiviral vector of SV40T and green fluorescent protein (GFP) fusion was constructed, and the multiplicity of infection (MOI\u0026thinsp;=\u0026thinsp;10) was optimized to achieve efficient transfection of sheep GCs (efficiency of 90%), and an immortalized cell line (GCs-SV40T-GFP) was established[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Preliminary experiments confirmed that the transfected cells remained stable in morphology and fluorescence expression after continuous passage to the 50th generation, which preliminarily verified the long-term integration and expression stability of SV40T gene[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAlthough SV40T immortalization technology has significant advantages, its application still faces two major challenges: one is the maintenance of cell function after immortalization[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. For example, immortalized human mesenchymal stem cells can retain the ability to differentiate, while some hepatocytes have reduced metabolic activity. For GCs, whether the synthesis ability of its core function-steroid hormones (such as estradiol) is impaired directly affects the research value of immortalized cells. The second is the potential tumorigenicity of SV40T[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e][\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Therefore, this study not only needs to verify the hormone secretion function of GCs-SV40T-GFP, but also needs to evaluate its biosafety through in vivo transplantation experiments[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAs an important economic animal and biomedical model, the reproductive physiology of sheep is of great significance to the development of animal husbandry breeding and human assisted reproductive technology. However, the short life characteristics of sheep GCs limit the depth of in vitro research[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In this study, an immortalized GCs-SV40T-GFP cell line was constructed to provide a stable and functional experimental model for analyzing the potential effects of follicular development mechanism, hormone regulation network and environmental pollutants on reproductive function in sheep[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In addition, if the model is proved to be safe and reliable, it can provide technical support for improving animal reproduction efficiency[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn summary, this study focused on SV40T lentiviral vector-mediated sheep granulosa cell immortalization technology, and systematically evaluated its proliferation characteristics, functional stability and biosafety, aiming to fill the technical gaps in this field and provide innovative tools for reproductive biology research and clinical application[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Treatment of GCs\u003c/h2\u003e\u003cp\u003eIn this study, 10 ovaries of 2-year-old healthy ewes were collected from the slaughterhouse in Tianzhu County, Gansu Province, and stored in 37\u0026deg; normal saline with penicillin and streptomycin (0.48g) added to the laboratory. Follicular fluid was extracted from ovarian follicles with a diameter of about 3\u0026thinsp;~\u0026thinsp;8 mm and injected into the tube. The filtrate was filtered with a 100 nm filter membrane, and then the follicular fluid was filtered with a 70 nm filter membrane, and then centrifuged with a high-speed centrifuge (TG16-WS desktop high-speed centrifuge) at 1500r/min to collect the precipitate. The cells were resuspended in DMEM medium containing 10% fetal bovine serum. Cell morphology was observed and cultured in a cell incubator at 37\u0026deg;C and 5% CO2.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Cell counting plate method\u003c/h2\u003e\u003cp\u003eIn this study, sheep granulosa cells were cultured in vitro, and the cell proliferation dynamics within 96 hours were systematically monitored by cell counting plate method. The specific method was as follows : synchronized granulosa cells were cultured in DMEM/F12 complete medium, and samples were taken every 12 hours from 0 h. Trypsin digestion and background trypan blue staining were used to count living cells under an inverted microscope using a modified Neubauer counting plate. Biological and technical repetitions were set at each time point. Finally, the cell density was calculated and the growth curve was drawn, and the population doubling time was calculated.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Immunofluorescence staining\u003c/h2\u003e\u003cp\u003eThe primary cultured GCs were seeded in 24-well plates, and the culture was terminated when the cells reached 80% confluence. Then immunofluorescence detection was performed. GCs were fixed with 4% paraformaldehyde for 30 min, and 0.1% Triton X-100 permeabilized the cell membrane for 15 min. It was blocked with 5% BSA (Gibco, Carlsbad, CA) at room temperature for 2 h. Cells were incubated with rabbit monoclonal antibody Vimentin at a concentration of 1:200 (Bioss, Beijing, China) overnight at 4\u0026deg;C. The cells were incubated with anti-rabbit secondary antibody at a concentration of 1:500 (Bioss, Beijing) and incubated at room temperature for 1 h the next day. Blank control was only incubated with secondary antibody. DAPI was used for nuclear re-staining. Cells were briefly washed three times with PBS between the start, end, and each step of staining. Immunostaining was evaluated by fluorescence microscopy. This experiment was repeated three times.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Confirmation of GFP-GCs (multiplicity of infection) in sheep granulosa cells\u003c/h2\u003e\u003cp\u003eOn the first day, SV40T sheep ovarian granulosa cells were inoculated in a 12-well plate containing 1E5 cells. The next day, the GFP virus stock solution ( Cegrogen Shanghai ) was taken out from the \u0026minus;\u0026thinsp;80\u0026deg;C refrigerator and melted in an ice bath before infection. The virus GFP stock solution was diluted with 800\u0026micro;L complete medium with MOI\u0026thinsp;=\u0026thinsp;1,10,50,100, and the original medium of the treatment group was removed. 500\u0026micro;L medium containing lentivirus dilution was added to the cells in the treatment group. The culture medium was changed on the third day, and the culture medium containing lentivirus was changed to 1 m L complete culture medium after 16 h of infection. On the 5th day, the infection efficiency was detected. The fluorescence was observed under an inverted fluorescence microscope, and the effect of lentivirus infection on target cells was calculated by fluorescence intensity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. SV40T-GFP-GCs lentivirus transfection\u003c/h2\u003e\u003cp\u003eOn the first day, SV40T sheep ovarian granulosa cells were inoculated in 12-well plates with 1E5 cells. The next day, before infection, the virus stock solution was taken out from the-80\u0026deg;C refrigerator and melted in an ice bath. The virus stock solution was diluted with 80\u0026micro;L complete medium at MOI\u0026thinsp;=\u0026thinsp;10, and the original medium of the treatment group was removed. The 500\u0026micro;L medium containing the lentivirus dilution was added to the cells of the treatment group. On the third day, the culture medium was replaced, and the culture medium containing lentivirus was replaced with 1 mL complete culture medium after 16 h of infection. On the 5th day, the infection efficiency was detected, and the fluorescence was observed under an inverted fluorescence microscope to determine the effect of lentivirus infection on target cells. The appropriate eukaryotic resistance screening cells were selected (the complete lethal concentration of puromycin was 3\u0026micro;g/mL, and the maintenance concentration was 1.5\u0026micro;g/mL). The cells were screened for two rounds of common drugs ( one round for 2 days, determined by antibiotic pre-experiment ). The cells could be stably passaged. After stable passage, DMEM\u0026thinsp;+\u0026thinsp;10% FBS\u0026thinsp;+\u0026thinsp;1%PS\u0026thinsp;+\u0026thinsp;1.5\u0026micro;g/mL puromycin complete medium was used to maintain the cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. RNA isolation, reverse transcription and quantitative RT-PCR.\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted using the Trizol method (SOLEIBO, Beijing, China), and the concentration and purity of RNA samples were evaluated using an ultra-micro UV-visible spectrophotometer (NanoReady/FC-1100) spectrophotometer. RNA samples with D260/D280 values between 1.8 and 2.0 were selected for cDNA synthesis using a PrimeTM TMRT kit (Takara, Beijing, China) with 1 \u0026micro;g of high-purity RNA. SYBR PreMix Ex Taq TM II (Takara, Beijing, China) and real-time PCR system (Bio-Rad, Hercules, CA, USA) were used for qRT-PCR. The reaction conditions are as follows: pre-denaturation at 95\u0026deg;C for 3 minutes, followed by denaturation at 95\u0026deg;C for 10 seconds, and annealing at 60\u0026deg;C for 30 seconds, for a total of 40 cycles. Each group consists of three biological replicates. The relative expression was calculated by 2-ΔΔCt. According to the gene sequence of sheep in GenBank database, primers were designed by Primer Premier 5.0 software for real-time fluorescence quantitative PCR. The primers were synthesized by Beijing Huada Gene Information Company (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eGCs-SV40T Real-time PCR primer sequences\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGene name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSequences (5\u0026rsquo;\u0026rarr;3\u0026rsquo;)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLength(bp)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAccession number\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCYP19A1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF-ATGCTGGTGCTGAGTATGTGGT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e192\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNM_001123000.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR-GCTGACAATCTTGAGGGTGTTG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eACTIN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF-ATATTGCTGCGCTCGTGGTT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e224\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNM_001009784.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR-GTTGGTGACAATGCCGTGCT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSV40T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF-TGGAAACCAAGTGCGACGAC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR-CGGCGAAGATAGCGGCATTA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eACTB-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF-ATTGCTGACAGGATGCAGAAGG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR-GCTGGAAGGTGGACAGTGAGG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Flow cytometry\u003c/h2\u003e\u003cp\u003eGCs-1, SV40T-GFP-GCs-1, SV40T-GFP-GCs-10, SV40T-GFP-GCs-25, SV40T-GFP-GCs-50 were added to 200\u0026micro;L cell suspension and added to 6-well plates. Add 10% fetal bovine serum complete medium, pre-incubated at 37\u0026deg;C, 5% CO2 incubator for 48 hours. Rise to about 90 percent, trypsin digestion and centrifugation. The stained cell suspension was transferred to a flow tube, and the data were detected and analyzed by flow cytometry.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. ELISA detection\u003c/h2\u003e\u003cp\u003eIn order to reduce the influence of fetal bovine serum, phenol red-free medium was used to avoid estrogen interference, and activated carbon/dextran-treated double-antibody FBS was replaced. Before the formal experiment, 24-hour serum starvation was performed and replaced with 0.5-1% stripped FBS medium. 28.6ng/m LA4 (androstenedione) was added. The primary, 10th, 25th and 50th generations were recorded respectively. When they grew to 100% of the cells, the complete medium was collected in a 1.5mL centrifuge tube. After centrifugation at 3000 rpm for 15minutes, the supernatant was taken. Concentrations were determined using kits (Shanghai, China, Mlbio).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9. West-blot assay\u003c/h2\u003e\u003cp\u003eThe treated cells were collected and lysate and PMSF (100:1, Servicebio, China) were added to the cell precipitate. Lysis was performed on ice for 30 min followed by centrifugation at 10,000\u0026times;g at 4℃for 15min. Protein concentration was determined using the BSA kit (Servicebio, China). The protein loading volume was 50mg. Separation was performed by electrophoresis using 10% SDS-PAGE followed by electrotransfer to polyvinylidene difluoride membranes (MCE, New Jersey, USA). The membranes were closed in 5% skim milk in TBS/Tween for 1 h at room temperature and then mixed with FHSR(Servicebio, China) 1:2000 and β-actin (Servicebio, China) 1:3000 with skim milk-PBST (Servicebio, China) and co-incubated. The membranes were then incubated with HRP conjugated antirabbit or anti-mouse secondary antibody 1:1000 (Servicebio, China) with 5% skim milk-PBST and detected by the Western Lighting ECL detection system. Signals were quantified using Image J 2020.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e2.10. Tumorigenicity experiment in mice\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eNine 5-week-old male BALB/c nude mice were purchased from Lanzhou Veterinary Research Institute and randomly divided into 3 groups with 3 mice in each group. After 1 week of adaptive culture, primary granulosa cells, 50th generation immortalized granulosa cells and HeLa cells were inoculated respectively. The cells were placed in two T25 culture flasks and cultured in DMEM medium containing 20% fetal bovine serum. The incubator was set at 37\u0026deg;C, 5% carbon dioxide. Until the cell growth is greater than 98%. After trypsin digestion, each generation of cells was adjusted to 1 \u0026times; 7 Ωcells / mL, and 10 \u0026micro;L was injected subcutaneously into the left back of mice. After 4 weeks of continuous observation, the tumor size was monitored with a ruler.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e2.11. Statistical analysis\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eAll data represent at least three independent experiments (with comparable results). ANOVA was used to evaluate the statistical significance of the data. The difference of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. One-way analysis of variance was used to analyze the data using GraphPad Prism 8 software, and paired comparisons were performed using LSD and Tukey tests. The results are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cb\u003eSuccessful isolation and identification of GCs cells.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe ovaries of healthy sheep were collected, stored at 37\u0026deg;C, and returned to the laboratory within 4 h. Follicular fluid was collected by puncturing follicles, and then follicular fluid was filtered with a 100u\u0026thinsp;+\u0026thinsp;70u filter. Primary culture was performed, and the first generation of cells was used for the experiment (Figure.1A). The cell growth density at different times was measured by cell counting (Figure.1B). The FSHR protein receptor was stained by immunofluorescence staining to verify that the cells were the required granulosa cells (Figure.1C).\u003c/p\u003e\u003cp\u003e\u003cb\u003eConfirmation of fluorescent GFP-GCs.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe GFP virus was used to determine the MOI, and the light expression efficiency of sheep ovarian granulosa cells reached 90% at MOI\u0026thinsp;=\u0026thinsp;10, and the pre-experiment passed. Subsequent experiments will be infected with MOI\u0026thinsp;=\u0026thinsp;10. When the second generation of cells grew to 80%, transfection was performed (P\u0026lt;0.05)(Fig.\u0026nbsp;2A,B).\u003c/p\u003e\u003cp\u003e\u003cb\u003eSV40T-GFP-GCs lentivirus was successfully transfected and the morphology was stable.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCell transfection was performed using SV40T-GFP virus with MOI\u0026thinsp;=\u0026thinsp;10. When the cells grew to 80%, SV40T-GFP transfection was performed (Fig.\u0026nbsp;3A). The transfection effect of SV40T-GFP-GCs was detected by q-PCR and fluorescence expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;3B). Cell growth was monitored by cell counting plate method (Figure.3C). The morphology of SV40T-GFP-GCs (primary, 1st, 10th, 25th and 50th generations) did not change, and the fluorescence was stably expressed, indicating that the SV40T-GFP gene could be stably expressed (Fig.\u0026nbsp;3D).\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe cell viability of SV40T-GFP-GCs was increased.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCell cycle was determined by flow cytometry. After the first generation, the S and G1 phase of the 10th generation of SV40T-GFP-GCs cells increased significantly. The S and G1 phases of the 25th and 50th generation of cells were also the same as those of GC (P\u0026lt;0.05)(Fig.\u0026nbsp;4A-C).\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe ability of SV40T-GFP-GCs cells to produce estradiol was enhanced.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe production of estradiol in SV40T-GFP-GCs cells after transfection was detected by ELISA (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;5A). Compared with the first generation of GCs-1, the estradiol content of SV40T-GFP-GCs-1 and SV40T-GFP-GCs-10 cells increased significantly, and the 25th and 50th generations were comparable to GCs-1 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;5B-C). The expression of CYP19a1 in SV40T-GFP-GCs was significantly enhanced by WB and q-pcr.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetection of SV40T-GFP-GCs cells do not have tumorigenicity.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGCs-1, SV40T-GFP-GCs-50, HELA cells were set as control (Fig.\u0026nbsp;6A,B). After 4 weeks of culture, the Hela cells had obvious tumors. GCs-1 cells and SV40T-GFP-GCs-50 cells had no tumors (Fig.\u0026nbsp;5C-E). Flow cytometry was used to detect the blood of mice, MDSC, PDL1, CD68 cells increased significantly (P\u0026lt;0.05)(Fig.\u0026nbsp;6F-H).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, an immortalized sheep granulosa cell line (GCs-SV40T-GFP) was successfully constructed by SV40T lentiviral vector, and its proliferation characteristics, functional stability and biosafety were systematically evaluated[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The experimental results are discussed from four aspects: technical advantages, function maintenance mechanism, security disputes and future application prospects[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, the transfection efficiency of SV40T gene mediated by lentiviral vector was as high as 90% (MOI\u0026thinsp;=\u0026thinsp;10), and the transfected cells still maintained stable morphology and fluorescence expression after continuous passage to the 50th generation[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. This result highlights the significant advantages of lentiviral vectors in gene delivery: their genomic integration ensures long-term expression of foreign genes, while low immunogenicity reduces interference with cell activity. Compared with traditional plasmid transfection or retrovirus, the ability of lentivirus to infect non-dividing cells further improves its application potential in primary cell immortalization. In addition, the introduction of GFP tags provides an intuitive tool for real-time monitoring of transfection efficiency and gene stability during cell passage. This design is innovative in similar studies[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIt is worth noting that SV40T significantly enhances the proliferation of GCs by targeting p53 and Rb proteins to relieve cell cycle arrest[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Cell cycle analysis showed that the proportion of S phase cells in GCs-SV40T-GFP increased, which was consistent with the results of human immortalized mesenchymal stem cells. However, the response of sheep GCs to SV40T may be species-specific[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. For example, the increase in cell proliferation rate in this study was higher than that in some bovine or mouse cell models, which may be related to the low original proliferation potential of sheep GCs. This finding suggests that the effect of SV40T immortalization is not only dependent on gene delivery efficiency, but also closely related to the biological characteristics of target cells[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAlthough immortalization is often accompanied by abnormal cell function, this study found that GCs-SV40T-GFP can continue to secrete estradiol, and the secretion level is not significantly different from that of primary cells. This result is consistent with the study of immortalized human luteal granulosa cells, indicating that SV40T may not interfere with steroidogenesis-related pathways (such as CYP19A1 expression or StAR protein activity)[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. It is further speculated that although the inhibition of p53 by SV40T may affect the DNA repair mechanism, it does not significantly destroy the core regulatory network of endocrine function of GCs. However, the potential impact of long-term passage on functional stability needs to be vigilant. For example, some immortalized hepatocytes have decreased metabolic enzyme activity after passage, which may be related to the accumulation of epigenetic modifications or mitochondrial dysfunction. Therefore, in the future, it is necessary to deeply analyze the global effect of SV40T on GCs signaling pathway in sheep through transcriptome or metabolomics analysis[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn addition, the secretion dynamics of other hormones (such as progesterone or inhibin) were not detected in this study. Sheep GCs have functional heterogeneity at different developmental stages of follicles, and whether immortalization causes cells to ' lock ' into specific functional states remains to be verified. If GCs-SV40T-GFP can still respond to gonadotropin (such as FSH) stimulation and regulate hormone synthesis, the model can be used to simulate the dynamic process of follicular development and has higher research value[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe risk of tumorigenicity of SV40T is the core issue that limits its clinical application. In this study, we confirmed that GCs-SV40T-GFP did not induce tumor formation by in vivo transplantation experiments in mice. This result contradicts some studies: for example, SV40T immortalized human fibroblasts can form sarcomas in immunodeficient mice[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The difference may be due to the following reasons: (1) Species specificity: the tumorigenic transformation threshold of sheep GCs may be higher than that of human cells; (2) Gene expression regulation: The promoter activity or copy number control of SV40T in lentiviral vector may reduce its carcinogenic potential. (3) Cell type difference: GCs themselves are terminally differentiated cells, and their malignant transformation requires additional driver mutations. Nevertheless, long-term safety still needs to be carefully evaluated. It is recommended that follow-up studies extend the observation period and use more sensitive detection methods (such as circulating tumor DNA analysis) to exclude the possibility of small lesions[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIt is worth noting that combined immortalization strategies (such as co-expression of SV40T and hTERT) have been shown to reduce genomic instability. If hTERT is introduced into sheep GCs, it may further optimize the genetic stability of the cell line and reduce the dose-dependent toxicity of SV40T[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe GCs-SV40T-GFP cell line constructed in this study provides an important tool for the study of sheep reproductive biology. For example, this model can be used to:(1) analyze the function of key genes for follicular development (such as FOXL2 or AMH) ; (2) Screening natural compounds or drugs that regulate the synthesis of estradiol ; (3) Assess the toxic effects of environmental pollutants (such as bisphenol A) on ovarian function. In addition, in the field of animal husbandry, this cell line can be used to optimize oocyte maturation conditions in in vitro fertilization (IVF) technology, or to develop a high-throughput screening platform for ovulation-promoting drugs. Compared with cattle or mouse models, data on GCs immortalization in sheep are still scarce. The success of this study provides a technical reference for the establishment of other large livestock germ cell models. For example, combined with CRISPR-Cas9 gene editing technology, specific gene knockout or mutation models can be constructed in immortalized cell lines to study the molecular mechanism of reproductive disorders.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThis study confirmed that SV40T lentiviral vector can efficiently realize the immortalization of sheep granulosa cells, and the immortalized cells meet the basic research needs in terms of proliferation ability, functional characteristics and biosafety. However, the following issues still need to be further explored:(1) The effect of immortalization on the epigenetic characteristics and stress response ability of GCs ;(2) The functional recovery efficiency of cell lines after long-term cryopreservation and resuscitation;(3) Synergistic effect of SV40T with other immortalized genes (such as hTERT). Future work can combine multi-omics technology with in vitro and in vivo functional verification to comprehensively evaluate the reliability of the cell line and expand its practical application in reproductive medicine and animal husbandry. In conclusion, the establishment of GCs-SV40T-GFP cell line not only fills the technical gap in the in vitro research model of sheep germ cells, but also lays a solid foundation for further exploring the regulation mechanism of follicular development and developing new reproductive intervention strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest regarding this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal ethics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental animal operations were performed in full accordance with institutional guidelines and approved by the Ethics Committee of the School of Life Sciences and Engineering of Northwest Minzu University (xbmu-sm-202593).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Natural Science Foundation of China [32460907], the Plateau Animal Disease Innovation Team [20240036], the Lanzhou Science and Technology Plan Project [No. 2022-2-\u0026shy;44]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eColour artwork\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eColour should be used only in online version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChen Hao\u003c/strong\u003e: designed the study, performed the experiments, and, Writing-original draft, the manuscript.\u003cstrong\u003e\u0026nbsp;Zhao Zhijie\u003c/strong\u003e: performed the experiments and, Writing-original draft, the manuscript.\u003cstrong\u003e\u0026nbsp;Ding Xiaona\u003c/strong\u003e: performed the experiments and, Writing-original draft, the manuscript.\u003cstrong\u003e\u0026nbsp;Li Pinsheng:\u003c/strong\u003e designed the study, performed the experiments, and, Writing-original draft, the manuscript.\u003cstrong\u003e\u0026nbsp;Zhao Bingzhu\u003c/strong\u003e: per-formed the experiments and, Writing-original draft, the manu-script. \u003cstrong\u003eYuan Yue\u003c/strong\u003e: performed the experiments and, Writing-original draft, the manuscript.\u0026nbsp;\u003cstrong\u003eRui Xiao\u003c/strong\u003e: designed the study, performed the experiments, and, Writing-original draft, the manuscript. \u003cstrong\u003eZhang Taojie\u003c/strong\u003e: hel-ped in performing the experiments and, Formal analysis, the, Data curation. \u003cstrong\u003eYingpai Zhaxi\u003c/strong\u003e: designed the study and helped in, Writing-original draft, the manuscript. \u003cstrong\u003eShengdong Huo\u003c/strong\u003e: designed the study and helped in, Writing-original draft, the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing nterest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDeclarations of interest: none.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThank Professor Huo Shengdong for his help.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHayakawa T. Establishment and characterization of immortalized sweat gland myoepithelial cells. Sci Rep 2022.\u003c/li\u003e\n\u003cli\u003eKwack MH, Hamida OB, Kim MK, Kim MK, Sung YK. Establishment and characterization of matched immortalized human frontal and occipital scalp dermal papilla cell lines from androgenetic alopecia. Sci Rep 2023;13:21421. https://doi.org/10.1038/s41598-023-48942-4.\u003c/li\u003e\n\u003cli\u003eMa T, Liu W, Jiang D, Zhang G, Zhao X, Zhang Y, et al. Analysis of Toxic Effects of Fluoride on Ovine Follicular Granulosa Cells Using RNA-Seq. Antioxidants 2024;13:506. https://doi.org/10.3390/antiox13050506.\u003c/li\u003e\n\u003cli\u003eZhang L, Liu K, Liu Z, Tao H, Fu X, Hou J, et al. In pre-clinical study fetal hypoxia caused autophagy and mitochondrial impairment in ovary granulosa cells mitigated by melatonin supplement. J Adv Res 2024;64:15\u0026ndash;30. https://doi.org/10.1016/j.jare.2023.11.008.\u003c/li\u003e\n\u003cli\u003eLin S. Establishment methods and research progress of livestock and poultry immortalized cell lines: A review. Front Vet Sci n.d.\u003c/li\u003e\n\u003cli\u003eMatsumura T, Takesue M, Westerman KA, Okitsu T, Sakaguchi M, Fukazawa T, et al. ESTABLISHMENT OF AN IMMORTALIZED HUMAN-LIVER ENDOTHELIAL CELL LINE WITH SV40T AND hTERT: Transplantation 2004;77:1357\u0026ndash;65. https://doi.org/10.1097/01.TP.0000124286.82961.7E.\u003c/li\u003e\n\u003cli\u003eTan J-J, Wang L, Mo T-T, Dai Y-F, Lu J, Liu X, et al. Establishment of Immortalized Laryngeal Epithelial Cells Transfected with Bmi1. Cell Transplant n.d.\u003c/li\u003e\n\u003cli\u003eWang J, Hu R, Wang Z, Guo Y, Wang S, Zou H, et al. Establishment of Immortalized Yak Ruminal Epithelial Cell Lines by Lentivirus-Mediated SV40T and hTERT Gene Transduction. Oxid Med Cell Longev 2022;2022:1\u0026ndash;17. https://doi.org/10.1155/2022/8128028.\u003c/li\u003e\n\u003cli\u003eZieliński M, Romanik-Chruścielewska A, Mikiewicz D, Łukasiewicz N, Sokołowska I, Antosik J, et al. Expression and purification of recombinant human insulin from E. coli 20 strain. Protein Expr Purif 2019;157:63\u0026ndash;9. https://doi.org/10.1016/j.pep.2019.02.002.\u003c/li\u003e\n\u003cli\u003eGuo L, Wang Z, Li J, Li J, Cui L, Dong J, et al. Immortalization effect of SV40T lentiviral vectors on canine corneal epithelial cells. BMC Vet Res 2022;18:181. https://doi.org/10.1186/s12917-022-03288-3.\u003c/li\u003e\n\u003cli\u003eSeridi N, Hamidouche M, Belmessabih N, El Kennani S, Gagnon J, Martinez G, et al. Immortalization of primary sheep embryo kidney cells. Vitro Cell Dev Biol - Anim 2021;57:76\u0026ndash;85. https://doi.org/10.1007/s11626-020-00520-y.\u003c/li\u003e\n\u003cli\u003eNorris N, Yau B, Kebede MA. Isolation and Proteomics of the Insulin Secretory Granule. Metabolites 2021;11:288. https://doi.org/10.3390/metabo11050288.\u003c/li\u003e\n\u003cli\u003eLi R-F, Nan G-X, Wang D, Gao C, Yang J, He T-C, et al. SV40T reprograms Schwann cells into stem-like cells that can re-differentiate into terminal nerve cells. Int J Dev Biol 2021;65:557\u0026ndash;62. https://doi.org/10.1387/ijdb.210062zz.\u003c/li\u003e\n\u003cli\u003eYang C, Lan W, Ye S, Zhu B, Fu Z. Transcriptomic Analyses Reveal the Protective Immune Regulation of Conjugated Linoleic Acids in Sheep Ruminal Epithelial Cells. Front Physiol 2020;11:588082. https://doi.org/10.3389/fphys.2020.588082.\u003c/li\u003e\n\u003cli\u003eLenz LS, Wink MR. The other side of the coin: mesenchymal stromal cell immortalization beyond evasion of senescence. Hum Cell 2023;36:1593\u0026ndash;603. https://doi.org/10.1007/s13577-023-00925-3.\u003c/li\u003e\n\u003cli\u003eHe H, Su X, Yang H, Zhang Y, Duan C, Yang R, et al. Effects of prolactin on the proliferation and hormone secretion of ovine granulosa cells in vitro. Anim Biosci 2024;37:1712\u0026ndash;25. https://doi.org/10.5713/ab.23.0448.\u003c/li\u003e\n\u003cli\u003eOshimura M, Tabata T, Uno N, Takata S, Hichiwa G, Kanazawa I, et al. Rejuvenation of human mesenchymal stem cells using a nonintegrative and conditionally removable Sendai virus vector. Sci Rep 2024;14:23623. https://doi.org/10.1038/s41598-024-74757-y.\u003c/li\u003e\n\u003cli\u003eZhan K, Yang TY, Chen Y, Jiang MC, Zhao GQ. Propionate enhances the expression of key genes involved in the gluconeogenic pathway in bovine intestinal epithelial cells. J Dairy Sci 2020;103:5514\u0026ndash;24. https://doi.org/10.3168/jds.2019-17309.\u003c/li\u003e\n\u003cli\u003eBruscoli S, Febo M, Riccardi C, Migliorati G. Glucocorticoid Therapy in Inflammatory Bowel Disease: Mechanisms and Clinical Practice. Front Immunol 2021;12:691480. https://doi.org/10.3389/fimmu.2021.691480.\u003c/li\u003e\n\u003cli\u003ePrimak A, Kalinina N, Skryabina M, Usachev V, Chechekhin V, Vigovskiy M, et al. Novel Immortalized Human Multipotent Mesenchymal Stromal Cell Line for Studying Hormonal Signaling. Int J Mol Sci 2024;25:2421. https://doi.org/10.3390/ijms25042421.\u003c/li\u003e\n\u003cli\u003eDos Santos A, Lyu N, Balayan A, Knight R, Zhuo KS, Sun Y, et al. Generation of Functional Immortalized Human Corneal Stromal Stem Cells. Int J Mol Sci 2022;23:13399. https://doi.org/10.3390/ijms232113399.\u003c/li\u003e\n\u003cli\u003eAlbericio G, Higuera M, Araque P, S\u0026aacute;nchez C, Herrero D, Garc\u0026iacute;a-Brenes MA, et al. Development of a Bmi1+ Cardiac Mouse Progenitor Immortalized Model to Unravel the Relationship with Its Protective Vascular Endothelial Niche. Int J Mol Sci 2024;25:8815. https://doi.org/10.3390/ijms25168815.\u003c/li\u003e\n\u003cli\u003eChen H, Li J, Pan X, Hu Z, Cai J, Xia Z, et al. A novel avian intestinal epithelial cell line: its characterization and exploration as an in vitro infection culture model for Eimeria species. Parasit Vectors 2024;17:25. https://doi.org/10.1186/s13071-023-06090-8.\u003c/li\u003e\n\u003cli\u003eLi J, Zhao B, Zhang X, Dai Y, Yang N, Bao Z, et al. Establishment and functional characterization of immortalized rabbit dermal papilla cell lines. Anim Biotechnol 2023:1\u0026ndash;10. https://doi.org/10.1080/10495398.2023.2252861.\u003c/li\u003e\n\u003cli\u003eChen H, Wang J, Zhao B, Yang Y, Yang C, Zhao Z, et al. N-Acetylcysteine relieving hydrogen peroxide-induced damage in granulosa cells of sheep. Cell Adhes Migr 2025;19:2484182. https://doi.org/10.1080/19336918.2025.2484182.\u003c/li\u003e\n\u003cli\u003eZhao S, Cui H, Fang X, Xia W, Tao C, Li J. Increased DNMT1 acetylation leads to global DNA methylation suppression in follicular granulosa cells during reproductive aging in mammals. BMC Genomics 2024;25:1030. https://doi.org/10.1186/s12864-024-10957-0.\u003c/li\u003e\n\u003cli\u003eHabara O, Logan CY, Kanai-Azuma M, Nusse R, Takase HM. WNT signaling in pre-granulosa cells is required for ovarian folliculogenesis and female fertility. Development 2021;148:dev198846. https://doi.org/10.1242/dev.198846.\u003c/li\u003e\n\u003cli\u003eTian H, Ren P, Liu K, Qiu C, Fan L, Li J, et al. Transcriptomic comparison of ovarian granulosa cells between adult sheep and prepubertal lambs. BMC Genomics 2022;23:151. https://doi.org/10.1186/s12864-022-08379-x.\u003c/li\u003e\n\u003cli\u003eMasumi S, Lee EB, Dilower I, Upadhyaya S, Chakravarthi VP, Fields PE, et al. The role of Kisspeptin signaling in Oocyte maturation. Front Endocrinol 2022;13:917464. https://doi.org/10.3389/fendo.2022.917464.\u003c/li\u003e\n\u003cli\u003eFang X, Xia W, Li S, Qi Y, Liu M, Yu Y, et al. SIRT2 Is Critical for Sheep Oocyte Maturation through Regulating Function of Surrounding Granulosa Cells. Int J Mol Sci 2022;23:5013. https://doi.org/10.3390/ijms23095013.\u003c/li\u003e\n\u003cli\u003eYang R, Duan C, Zhang S, Liu Y, Zhang Y. Prolactin Regulates Ovine Ovarian Granulosa Cell Apoptosis by Affecting the Expression of MAPK12 Gene. Int J Mol Sci 2023;24:10269. https://doi.org/10.3390/ijms241210269.\u003c/li\u003e\n\u003cli\u003eAbdurahman A, Aierken W, Zhang F, Obulkasim R, Aniwashi J, Sulayman A. miR-1306 induces cell apoptosis by targeting BMPR1B gene in the ovine granulosa cells. Front Genet 2022;13:989912. https://doi.org/10.3389/fgene.2022.989912.\u003c/li\u003e\n\u003cli\u003eZhai B, Li X, Zhao Z, Cao Y, Liu X, Liu Z, et al. Melatonin Protects the Apoptosis of Sheep Granulosa Cells by Suppressing Oxidative Stress via MAP3K8 and FOS Pathway. Genes 2023;14:1067. https://doi.org/10.3390/genes14051067.\u003c/li\u003e\n\u003cli\u003eDai T, Kang X, Yang C, Mei S, Wei S, Guo X, et al. Integrative Analysis of miRNA-mRNA in Ovarian Granulosa Cells Treated with Kisspeptin in Tan Sheep. Animals 2022;12:2989. https://doi.org/10.3390/ani12212989. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"SV40T lentiviral vector, granulosa cells, immortalization","lastPublishedDoi":"10.21203/rs.3.rs-7555331/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7555331/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eFollicular granulosa cells are an important cell source for studying animal reproductive function.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThe aim of this study was to investigate the gene transfection of sheep ovarian granulosa cells (GC) mediated by SV40 T lentiviral vector. The effects of GC immortalization and functional properties were verified. The results showed that the cell line could be continuously passaged to the 50th generation. And the cell morphological characteristics and fluorescence expression intensity remained stable. By optimizing the infection conditions (MOI\u0026thinsp;=\u0026thinsp;10), efficient transfection of GFP-tagged SV40T gene was successfully achieved (efficiency of about 90%)(P\u0026lt;0.05). The immortalized sheep granulosa cell line (GCs-SV40T-GFP) was successfully established. The growth rate of immortalized cells was significantly higher than that of primary GCs-1 cells. Flow cytometry showed that the proportion of S phase and G1 phase of immortalized cells increased significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). WB and q-pcr confirmed that the transfected cells continued to secrete estradiol to maintain endocrine function (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, in vivo safety evaluation showed that GCs-SV40T-GFP cells did not induce mouse tumors.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eIn this study, a functionally stable immortalized sheep granulosa cell model was successfully constructed. At the same time, it also provides an important tool for animal reproductive function research and hormone regulation mechanism exploration.\u003c/p\u003e","manuscriptTitle":"Establishment and identification of immortalized sheep ovarian granulosa cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-25 13:20:09","doi":"10.21203/rs.3.rs-7555331/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-23T09:51:18+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-21T22:20:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"257301371502064297735102928098360085289","date":"2025-10-10T08:14:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-05T18:50:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"124134182958502583626521650366771104448","date":"2025-09-25T11:25:57+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-16T14:46:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-16T14:40:23+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-15T06:47:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-12T08:23:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Veterinary Research","date":"2025-09-12T08:19:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ac52c2f3-6bc3-46d7-b151-1288936ff488","owner":[],"postedDate":"September 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T16:01:35+00:00","versionOfRecord":{"articleIdentity":"rs-7555331","link":"https://doi.org/10.1186/s12917-026-05472-1","journal":{"identity":"bmc-veterinary-research","isVorOnly":false,"title":"BMC Veterinary Research"},"publishedOn":"2026-04-21 15:57:40","publishedOnDateReadable":"April 21st, 2026"},"versionCreatedAt":"2025-09-25 13:20:09","video":"","vorDoi":"10.1186/s12917-026-05472-1","vorDoiUrl":"https://doi.org/10.1186/s12917-026-05472-1","workflowStages":[]},"version":"v1","identity":"rs-7555331","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7555331","identity":"rs-7555331","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}