WO2020019522A1

WO2020019522A1 - Conditioned cell immortalization lentiviral vector and construction method thereof, and application thereof in establishment of porcine granulosa cell line

Info

Publication number: WO2020019522A1
Application number: PCT/CN2018/108950
Authority: WO
Inventors: 白银山; 朱翠; 冯美莹; 张守全; 詹小舒; 李巨浪
Original assignee: 佛山科学技术学院
Priority date: 2018-07-25
Filing date: 2018-09-30
Publication date: 2020-01-30
Also published as: CN109022488A

Abstract

Provided are a construction method of a lentiviral vector for conditioned immortalization of a cell, a pLVX-Tet3G-Large T-T2A-mCherry-Puro ^r lentivirus obtained by packaging by using the lentiviral vector and an application thereof in establishment of a porcine granulosa cell line. A reversible and inducible porcine granulosacell line is established by means of a Tet-on-3G system; when Dox is added, the cell line can stably maintain cell proliferation in vitro; when Dox is removed, the granulosa cell can be differentiated into a functional terminal cell to secrete more estrogens and progesterones, but loses proliferation ability; and when Dox is added again, the cell can restore to a proliferation state.

Description

Conditional cell immortalized lentiviral vector, construction method thereof and application in porcine ovarian granulocyte cell line construction

Technical field

The invention relates to the field of genetic bioengineering, in particular to a lentiviral vector, a method for constructing the same, and application in a porcine ovary granular cell line.

Background technique

Granulosa cells (GCs) are a group of important cells in the follicle that secrete estrogen and progesterone, and play an important role in regulating oocyte development, ovulation and the maintenance of pregnancy. The newly isolated GCs cultured in vitro are of great value in studying the application of hormone synthesis and oocyte development in female animals. At the same time, they also provide a basis for the identification of methods for the diagnosis and treatment of female reproductive diseases and the implementation of techniques to reduce reproductive losses in livestock. stand by. However, GCs cells cultured in vitro are easy to differentiate and have limited proliferation potential, which limits their application and research.

Many research results show that long-term culture of primary cells is usually established by transferring exogenous LargeT or telomerase reverse transcriptase (Tert). LargeT can promote cell proliferation and anti-aging by inhibiting cell cycle inhibitors P53 and pRb. To make cells immortal. Establishing immortal GCs lines, although retaining the main GCs gene expression pattern and some functional characteristics, but lost the differentiation function and characteristics of many primary cells.

Previous studies have shown that many granulosa cells have been established in pigs, and 6 granulosa cell lines (named MDG2.1, PGC-2, jc-410, CG-9, PGV3, AVG-16) have been reported in the article, but the current market There is still a lack of available granule cell lines. These reported granule cell lines are not ideal cell models to study porcine GCs function because these cell lines lose the response to follicle stimulating hormone (FSH) and luteinizing hormone (LH) stimulation to promote steroid hormones. Such as the secretion of estradiol (E2) and progesterone (P4), can not produce inhibin and in vitro differentiation, and there are great differences compared with primary cells. In fact, most cell lines lose their physiological functions due to integration of Large T or Tert into the genome, which is far from the physiological functions of primary cells.

In recent years, research on conditional gene expression has received great attention. Some studies have tried to remove the Large T or Tert gene through the "Cre / loxps" method to obtain differentiated functional cells, with success. The present invention successfully applies the Tet-on-3G method to establish a reversible and inducible porcine ovarian granulosa cell line.

Summary of the Invention

The purpose of the present invention is to solve the above-mentioned shortcomings of the prior art and to use a lentivirus transgene method to establish a reversible and inducible porcine ovarian granulosa cell line through the Tet-on-3G system, which can both induce in vitro cell proliferation and can Restores the characteristics of in vitro cell differentiation.

A technical solution adopted by the present invention is to provide a method for constructing a conditional cell immortalized lentiviral vector, which includes the following steps:

1) According to molecular cloning technology, Tet-on-3G lentiviral vector was double-digested by Nhe1 and Bamh1, and a 7595bp vector fragment was recovered;

2) Using

primers

1 and 2 and pLVX-Large T-EF1-EGFP as a template to obtain a Large T-T2A fragment, the sequences of primer 1 and primer 2 are shown in SEQ ID NO1 and 2; using primer 3 And primer 4, using pLVX-sgRNA-mCherry as a template to obtain a T2A-mCherry fragment, the sequences of primer 3 and primer 4 are shown in SEQ ID NO 3 and 4;

3) Use the Large T-T2A fragment and T2A-mCherry fragment obtained in step 2) as templates, add primers 1 and 4, and perform overlapping PCR amplification to obtain a DNA fragment of LargeT-T2A-mCHRRY, and perform double enzymes through Nhe1 and Bamh1 Cut and recycle

4) Linking the LargeT-T2A-mCHRRY obtained in step 3) into the Tet-on-3G lentiviral vector through T4 ligase, and constructing and obtaining pLVX-Tet3G-Large T-T2A-mCherry-Puro ^r lentivirus.

Another object of the present invention is to provide the application of the pLVX-Tet3G-Large T-T2A-mCherry-Puro ^r lentivirus as described above in the establishment of porcine ovarian granulocyte cell lines.

Another technical solution adopted by the present invention is: a method for constructing a reversible and inducible porcine ovarian granulosa cell line, comprising the steps:

1) Collect porcine ovarian granulosa cells and expand the culture to obtain primary porcine ovarian granulosa cells to be infected;

2) 293FT cell plating, when the cell density reaches 70-80%, perform liquid exchange, add 7 mL of complete antibiotic-free medium containing 10% FBS as the cell culture medium, and prepare for virus packaging;

3) Pack the lentivirus according to the virus packaging method to obtain pLVX-Tet3G-Large T-T2A-mCherry-Puro ^r lentivirus;

4) Take 10 μg of step 3) The obtained pLVX-Tet3G-Large T-T2A-mCherry-Puro ^r lentivirus is mixed with 3.5 μg PMD2.G, 10.4 μg psPAX2, 80 μL Polyfect transfection reagent and 300 μL DMEM basic medium. The lentiviral packaging plasmid transfection mixed solution was obtained, and then the lentiviral packaging plasmid transfection mixed solution was added dropwise to the cell culture dish obtained in step 2) for transfection, and the virus-containing supernatant was collected at 48h and 72h after transfection. Clear, filter, and centrifuge to obtain pLVX-Tet3G-Large T-T2A-mCherry-Puro ^r virus solution;

5) According to the lentiviral manual, the primary porcine ovarian granulosa cells obtained in step 1) were infected with Polyberene transfection enhancer and the pLVX-Tet3G-Large T-T2A-mCherry-Puro ^r virus solution obtained in step 4), Dox induction was added at 24 hours, and puromycin was added at 72 hours for screening. Two weeks later, a stable reversible and inducible porcine ovarian granulosa cell line was obtained.

The beneficial effects of the present invention are:

For the first time, the present invention establishes a reversible and inducible porcine ovarian granulosa cell line through the Tet-on-3G system. The cell line has the characteristics of inducibility and reversal. When Dox is added, it can stably maintain cell proliferation in vitro; when Dox is removed, ovarian granulosa cells can restore the function of in vitro differentiation, produce functional cells, secrete more estrogen and progesterone, and then lose the proliferation ability; when added again Upon Dox, the cells will return to a proliferative state. The reversible cell line is of great significance for studying cell proliferation, development and dedifferentiation. At the same time, according to the biological properties of ovarian granulocytes, the cell line of the present invention has important applications for studying ovarian granulocyte hormone secretion and regulation of oocyte development. Value, at the same time in the development of breeding-related technologies and application prospects for drug or vaccine research and development.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the pLVX-Tet3G-Large T-T2A-mCherry-Puro ^r lentiviral map;

Figure 2 is a cell micromorphology (A, primary cultured GCs; B and C, lentiviral transfection to obtain stable expression of Large T and mCherry GCs; D and E, addition of Dox induced GCs to stable expression of Large T and mCherry , In vitro continuous culture for more than 6 months; F, non-transgenic primary GCs cells after 5 days in vitro culture);

Figure 3 shows the mCherry expression of GCs without Dox (A-D: 24, 42, 48, and 96h fluorescence microscope observation of GCs cells mCherry expression without Dox);

Figure 4 shows the detection of cell proliferation (A, GCs morphological changes, obvious changes in cell morphology without the addition of Dox culture; B, cell proliferation curve of continuous culture for 8 days with and without the addition of Dox; C, cell cycle Analysis shows that during Dox induction, S / M phase cells increase, and in the absence of Dox, the percentage of S / M phase cells decreases);

FIG. 5 is an immunofluorescence detection chart of characteristic proteins of GC cells;

Figure 6 shows GC cell hormone secretion detection (A, Western blot analysis of gonadotropin receptor and steroid hormone synthetase expression; B and C, respectively, estradiol and progesterone with and without Dox addition The data are the average of 6 experiments, * p <0.05);

Figure 7 is a micrograph of the regulation of GCs dedifferentiation (A, GCs cells cultured for 14 days without Dox; B, GCs cultured for 1 day after adding Dox; C and D, GCs cultured for 2 days with Dox; E and F, continue GCs were induced for 9 days).

detailed description

The following describes the present invention in detail with reference to the embodiments, so that those skilled in the art can understand the present invention. It is necessary to specifically point out that the embodiments are only for further explanation of the present invention, and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make non-essential improvements and improvements to the present invention based on the above contents. Adjustment should still belong to the protection scope of the present invention. At the same time, the raw materials mentioned below are all commercially available products; the process steps or preparation methods not mentioned in detail are all process steps or preparation methods known to those skilled in the art.

In the present invention, the Large T and mCherry genes are designed to construct a Tet-on-3G vector that is jointly controlled by the P-TRE-3G promoter. The two genes are connected by a self-shearing sequence T2A, and are stabilized by a lentiviral transgene method Integrated into the genome of ovarian granulosa cells (hereinafter referred to as GCs).

Under the condition of Dox culture, cells express Large T gene and red fluorescent protein mCherry. GCs rapidly and stably proliferate in vitro. This cell line was continuously cultured for more than 6 months in vitro. Red fluorescence detection showed that mCherry-positive cells reached 100%. When Dox was added, the cells began to significantly reduce mCherry expression at 24h, very few mCherry-expressing cells could be observed at 42h, and no more mCherry expression after 48h; the cells also had morphological changes at the same time, and began to grow on the fourth day of culture The morphology became longer and larger on day 6 of culture.

Using flow cytometry, it was found that without the addition of Dox cells, the proliferative capacity has gradually been lost. From the second day of culture, the proportion of S / G2 phase cells has been greatly reduced. By the sixth day of culture, the cells in the S phase are almost 0. ; The growth curve also showed that the proliferative capacity of the cells without Dox particles gradually decreased, and the number began to decrease significantly on the 8th day of culture.

Immunofluorescence test results showed that granule cell-specific proteins (FSHr, CYP11A1, CYP17A1, and 3β-HSD) were expressed in cells with and without Dox, and these proteins could promote the synthesis of hormones by cells. Western-blot test results showed that the steroid metabolism-related enzymes CYP11A1, CYP17A1, and 3β-HSD were significantly increased on the 6th day without Dox culture, while the expression levels of the dry markers LIFr and FSHr were reduced, which combined with the morphological analysis of the cells indicated that It has been differentiated. Using hormone detection ELISA kit to analyze cells cultured on

days

2, 4, and 6 with or without Dox culture, it is found that there is a significant difference in the level of hormone secretion, and the secretion gradually increases with time. The increase indicates that the cells have differentiated.

Finally, the number of cells cultured without Dox was significantly reduced after 8 days of culture. By 14 days, most of the cells had died, and only the cells with poor viability and morphology remained. At this time, the addition of Dox induced these cells to express Large T again. It was found that after 2 days of culture, these cells expressed fluorescence again, while their morphology returned to short and well refracted. After 9 days of culture, the cells were full again and expressed mCherry, which indicates that the present invention has successfully established an inducible, differentiated and Reversible cell lines have important application value for studying granulocyte hormone secretion and oocyte development, and are used in the development of reproduction-related technologies and in the development of drugs or vaccines.

Example 1

A method for constructing a conditional cell immortalized lentiviral vector, comprising the steps of:

1) According to molecular cloning technology, Tet-on-3G lentiviral vector (Addgene, 50661) was double-digested by Nhe1 and Bamh1, and a 7595bp vector fragment was recovered;

2) Using primer 1 (sequence is shown in SEQ ID NO1) and primer 2 (sequence is shown in SEQ ID NO2), using pLVX-Large T-EF1-EGFP as a template to obtain a Large T-T2A fragment with a length of 2155bp ; Using primer 3 (sequence is shown in SEQ ID NO3) and primer 4 (sequence is shown in SEQ ID NO4), using pLVX-sgRNA-mCherry as a template to obtain a T2A-mCherry fragment, 774bp in length;

3) Use the Large T-T2A fragment and T2A-mCherry fragment obtained in step 2) as templates, add primers 1 and 4 and perform overlapping PCR amplification to obtain a DNA fragment with a length of 2875 bp of Large T-T2A-mCHRRY fragment;

4) Digestion with Nhe1 and Bamh1 and recovery.

5) Link TL-T2A-mCHRRY into the Tet-on-3G lentiviral vector through T4 ligase, that is, construct pLVX-Tet3G-Large T-T2A-mCherry-Puro ^r lentivirus, as shown in Figure 1.

Example 2

A method for constructing a reversible and inducible porcine ovarian granulosa cell line, comprising the steps of:

1) Isolation and culture of granular cells:

Swine ovary samples were taken from a commercial slaughterhouse and brought back to the laboratory in sterile physiological saline (38 ° C) at 2 h internal temperature and then transferred to the laboratory. Use a 10mL syringe to aspirate the oocytes and ovarian granulosa cells in the follicular fluid, and then remove the oocytes and the complexes of the oocytes and granulocytes with a mouth pipette to collect porcine ovarian granulosa cells, centrifuge at 600g for 5min, collect the cells, and wash Three times, cultured in a 10 cm ² petri dish, the culture system was DMEM (Gibco, 11960044)) + 10% fetal bovine serum (Gibco, 10099-141) + 1% GlutaMAX ^TM (Gibco, 35050061) + 20ng / mL epidermal growth Factor (Peprotech, AF-100-15) to obtain primary porcine ovarian granulosa cells;

2) Lentiviral packaging and cell infection and screening:

a, Add 300 μL of DMEM basal medium to a 1.5 mL centrifuge tube, add 3.5 μg of PMD2.G, 10.4 μg of pspax2, 10 μg of pLVX-Tet3G-Large T-T2A-mCherry-Puro ^r lentivirus and 80 μL Polyfect transfection reagent, mix gently and let stand for 5-10 minutes, equipped with lentivirus packaging plasmid transfection mixture;

b, 293FT cells were plated 1 day in advance of antibiotic-free DMEM (containing 10% FBS) Cells were plated in 10cm ² petri dish per plate at 10 ⁵ cells / mL, seeded cells, when the cell density reached 70-80% At that time, the 293FT cells of each plate were carefully changed, and washed gently with PBS, and 7 mL of complete antibiotic-free medium containing 10% FBS was added to obtain a cell culture solution;

c. Add the lentiviral packaging plasmid transfection mixture evenly to 1 mL of complete antibiotic-free medium, mix thoroughly and add it evenly to the cell culture solution, change the solution 5 to 6 hours after transfection, and add 8 mL of The complete antibiotic medium was continued to culture. After 24 hours of transfection, the transfection was observed under a fluorescence microscope and the cells were exchanged in full. After 8 hours, the virus-containing supernatant was harvested into 15 mL centrifuge tubes, centrifuged at 3000 rpm for 3 minutes, and filtered through a 0.45 μm filter. The filtrate was stored at 4 ° C, waiting for ultracentrifugation. At the same time, 10 mL of complete culture medium was added to the Petri dish to continue culturing the cells. After 72 hours, the virus-containing supernatant was harvested again into a 15 mL centrifuge tube, centrifuged at 3000 rpm for 3 minutes, and filtered with a 0.45 μm filter. Store at 4 ℃ and wait for ultracentrifugation to obtain pLVX-Tet3G-Large T-T2A-mCherry-Puro ^r virus solution;

d. According to the lentiviral manual, take the cultured primary porcine ovarian granulocytes and calculate based on the MOI value = 10, add 6 μg / mL Polybrene transfection enhancer and the pLVX-Tet3G-Large T-T2A-mCherry obtained above -Puro ^r virus solution for virus infection, 6h change of solution, 24h with Dox induction, observe the fluorescence after 72h and judge the cell infection, if the fluorescence ratio is low, perform a second infection treatment, after most cells express mCherry, 1 μg / mL puromycin (Gibco, A1113803) was added for screening. Two weeks later, a stable reversible and inducible porcine ovarian granulosa cell line was obtained.

Example 3: Isolation of GCs and Infection of Large T Gene

The morphology of the primary isolated and cultured GCs of the present invention is short and fibrous (as shown in FIG. 2A). It was transfected with pLVX-Tet3G-Large T-T2A-mCherry-Puro ^r lentivirus and added with Dox to induce success. GCs cell transgenes were obtained (as shown in Figures 2B and 2C); Large T gene was successfully transfected by puromycin selection, and the cell positive rate reached 100% mCherry expression, while maintaining fibroblast morphology and rapid proliferation, continuous in vitro After being cultured for more than 6 months (as shown in Figs. 2D and 2E), the untransformed primary cells were cultured to the 5th day, and the morphology began to change significantly, and the refractive index and state became worse (as shown in Fig. 2F).

Example 4: Time-point detection of Large T missing expression when Dox is not added

Observe the expression of mCherry through a fluorescence microscope. Set 4 time points. The detection result of Larget T begins to decrease significantly at 24h (as shown in Figure 3A). Only a small part of GCs at 42h indicate very weak mCherry fluorescence (such as Figure 3B); at 48h, almost all GCs did not observe mCHRRY expression (as shown in Figure 3C), and in the other 48h, GCs began to show morphological changes, and mCherry (such as (Figure 3D).

Example 5: Detection of GCs Proliferated by Dox

There was a significant difference between GCs with and without Dox. The morphology changed significantly on the 4th day without Dox. The morphology began to pull up and became larger. By the 6th day, the morphology had become very long. The number was significantly smaller than that in the Dox group (as shown in Figure 4A); the cell proliferation curve showed that the proliferation ability of the cells without Dox particles significantly decreased, and the number began to decrease significantly on the 8th day of culture (as shown in Figure 4B); Using flow cytometry, it was found that without the addition of Dox cells, the proliferation ability has gradually been lost. From the second day of culture, the proportion of S / G2 phase cells has decreased significantly. By the sixth day of culture, the cells in the S phase are almost 0. (As shown in Figure 4C).

Example 6: Immunofluorescence detection of characteristic proteins of GC cells

As shown in Figure 5, FSHr, CYP11A1, CYP17A1, and β-HSD were detected by immunofluorescence in GCs with and without Dox, indicating that both types of cells are GCs.

Example 7: Analysis of GC cell hormone synthesis amount

Western-blot protein detection

1) GCs protein extraction: collect two sets of proteins with and without Dox, wash them twice with PBS, then add RIPA lysate (containing 110 μL / mL PMSF and 5 μL / mL protease inhibitor), lyse on ice for 30 min, each Shake every 5 minutes, centrifuge at 12,000 rpm, 4 ° C and collect the supernatant;

2) Determination of protein concentration: The BCA protein quantification kit is used for measurement. For detailed operations, refer to the instructions. Based on the measured protein concentration, use RIPA lysate to adjust the protein concentration of the sample to the same, and store it in a refrigerator at -80 ° C after dispensing.

3) SDS-PAGE protein electrophoresis: prepare 10% separation gel and 5% concentrated gel, perform SDS denaturation treatment before protein loading, lyse the sample, add 5 × SDS loading buffer at 4: 1, and shake at 95 ° C for 5 min. The sample volume is 20 μg; the electrophoresis conditions are: constant voltage 80V, 40min, 120V, 1h;

4) Transfer film: constant current 250mA-2.5h, wet transfer on ice;

5) Blocking: Block at room temperature for 30 min in 3% BSA solution;

6) Incubate primary antibodies (Rabbit anti-LIFr, Bioss-bs-1458R, Rabbit anti-FSHr, Absin-abs120271; Rabbit anti-CYP11A1, Liankebio-AB2060; Rabbit anti-CYP17A1, Liankebio-AB1766; Rabbit antianti-3β-HSD , Abcam-ab150384) The incubation antibody was diluted with blocking solution. After overnight incubation at 4 ° C, it was washed with 1 × TBST for 4 × 5min / times.

7) Incubate secondary antibody (1: 2,000, Santa Cruz; SC-2030). Incubate: Dilute the corresponding IgG with blocking solution at 1: 5000, incubate for 1 h at room temperature, and wash with 1 × TBST for 4 × 5 min / times.

8) Development and gray scale analysis: ECL chemiluminescence reagents are used for imaging and development in a chemiluminescence gel imaging system, and Imagelab (American Bio-Rad) software is used for image editing.

Hormone level test

1) Inoculate GCs cells in 24-well plates, add Dox-free and Dox-free cultures, set up 6 replicates per group, and collect the culture supernatant at 2, 4, and 6 days of culture, respectively;

2) Detection and analysis according to the instructions of the ELISA kit, porcine estradiol ELISA Kit (Mlbio, ML-79414, China) and porcine progesterone ELISA Kit (Mlbio, ML-79329, China).

Western-blot test results showed that the steroid metabolism-related enzymes CYP11A1, CYP17A1 and 3β-HSD were significantly increased on the 6th day without Dox culture, while the dry marker LIFr and FSHr expressions were decreased (as shown in Figure 6A). Morphological analysis of the cells indicates that the cells have differentiated. Analysis of cells cultured on

days

2, 4, and 6 using the ELISA kit for hormone detection, with and without the addition of Dox, found that the levels of hormones (E2 and P4) were significantly increased. There are significant differences between the two groups, and the amount of secretion gradually increases over time, indicating that the cells have differentiated (as shown in Figures 6B and 6C).

Example 8: Regulation of dedifferentiation of differentiated GCs cells

The number of cells cultured without adding Dox decreased significantly after 8 days of culture, and most of them had died by 14 days. The only cells with poor viability and morphology were left (as shown in Figure 7A). At this time we added Dox to induce these again. The cells expressed Large T. The results showed that the morphology of these cells began to change after one day of culture, began to shorten, and the refractive index began to increase (as shown in Figure 7B). After two days of culture, the cells began to express fluorescent proteins and the morphology returned to Short and very refractive (as shown in Figures 7C and 7D), after 9 days of culture, the cells were full and all expressed fluorescent proteins (as shown in Figures 7E and 7).

The above embodiment is a preferred embodiment of the present invention, and all processes similar to the present invention and equivalent changes made by the present invention shall belong to the protection scope of the present invention.

Claims

A method for constructing a conditional cell immortalized lentivirus vector, comprising the steps of:

1) According to molecular cloning technology, double-digestion of the Tet-on-3G lentiviral vector to recover a 7595bp vector fragment;

2) Using primers 1 and 2 and pLVX-Large T-EF1-EGFP as a template to obtain a Large T-T2A fragment, the sequences of primer 1 and primer 2 are shown in SEQ ID NO1 and 2; using primer 3 And primer 4, using pLVX-sgRNA-mCherry as a template to obtain a T2A-mCherry fragment, the sequences of primer 3 and primer 4 are shown in SEQ ID NO 3 and 4;

3) Use the Large T-T2A fragment and T2A-mCherry fragment obtained in step 2) as templates, add primers 1 and 4, and perform overlapping PCR amplification to obtain a LargeT-T2A-mCHRRY DNA fragment, double-digest and recover;

4) Ligating the LargeT-T2A-mCHRRY obtained in step 3) into the Tet-on-3G lentiviral vector through ligase, and constructing and obtaining pLVX-Tet3G-Large T-T2A-mCherry-Puro r lentivirus.
The method for constructing a lentiviral vector according to claim 1, wherein the double-enzyme digestion in step 1) and step 3) is double-enzyme digestion by Nhe1 and Bamh1.
The method for constructing a conditional cell immortalized lentiviral vector according to claim 1, wherein the ligase in step 4) is T4 ligase.
A pLVX-Tet3G-Large T-T2A-mCherry-Puro r lentivirus obtained by the method for constructing a conditional cell immortalized lentiviral vector according to any one of claims 1 to 3.
The use of pLVX-Tet3G-Large T-T2A-mCherry-Puro r lentivirus according to claim 4 in the establishment of porcine ovarian granulocyte cell lines.
A method for constructing a reversible and inducible porcine ovarian granulosa cell line, comprising the steps of:

1) Collect porcine ovarian granulosa cells and expand the culture to obtain primary porcine ovarian granulosa cells to be infected;

2) 293FT cell plating, when the cell density reaches 70-80%, perform liquid exchange, add 7 mL of complete antibiotic-free medium containing 10% FBS as the cell culture medium, and prepare for virus packaging;

3) Pack the lentivirus according to the virus packaging method to obtain pLVX-Tet3G-Large T-T2A-mCherry-Puro r lentivirus;

4) The pLVX-Tet3G-Large T-T2A-mCherry-Puro r lentivirus obtained in step 3) is mixed with Polyfect transfection reagent and DMEM to obtain a lentiviral packaging plasmid transfection mixture, and the lentiviral packaging plasmid is transfected The mixed solution was added dropwise to the cell culture solution obtained in step 2) for transfection, and the supernatant containing the virus was collected at 48h and 72h of transfection, filtered, and centrifuged to obtain pLVX-Tet3G-Large T-T2A-mCherry-Puro r Viral fluid

5) According to the lentiviral instruction manual, the primary porcine ovarian granulosa cells obtained in step 1) were infected with pLVX-Tet3G-Large T-T2A-mCherry-Puro r virus solution obtained in step 4). Dox induction was added for 24 hours and 72 hours. Puromycin was then added for screening. Two weeks later, stable reversible and inducible porcine ovarian granulosa cell lines were obtained.
The method for constructing a reversible porcine ovary cell line according to claim 6, characterized in that: the lentiviral packaging plasmid transfection mixed solution of step 2) comprises 3.5 μg PMD2.G, 10.4 μg psPAX2, 10 μg of pLVX-Tet3G-Large T-T2A-mCherry-Puro r lentivirus, 80 μL of Polyfect transfection reagent, and 300 μL of DMEM basal medium.
The method for constructing a reversible porcine ovarian cell line according to claim 6, characterized in that: in step 5), a polyberene transfection enhancer is added to assist virus infection.