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

A technology of a lentiviral vector and a construction method, which is applied to the lentiviral vector and its construction method and the application field in the porcine ovary granulosa cell line, can solve the problems of difference, lack of granulosa cell line, inability to produce inhibin and in vitro differentiation, etc.

Pending Publication Date: 2018-12-18
FOSHAN UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Previous studies have shown that many pig granulosa cells have been established, and 6 granulosa cell lines (named MDG2.1, PGC-2, jc-410, CG-9, PGV3, AVG-16) have been reported, but the current market There is still a lack of granulosa cell lines available on the Internet, and these reported granulosa cell lines are not ideal cell models to study the function of porcine GCs, because these cell lines lose the ability to respond to follicle-stimulating hormone (FSH), luteinizing hormone (LH) stimulation and promote steroid hormones. , such as the secretion of estradiol (E2) and progesterone (P4), can not produce inhibin and differentiate in vitro, and there is a big difference compared with primary cells

Method used

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

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Embodiment 1

[0035] A method for constructing a conditional cell immortalized lentiviral vector, comprising the steps of:

[0036] 1) According to the molecular cloning technique, the Tet-on-3G lentiviral vector (Addgene, 50661) was double digested with Nhe1 and Bamh1, and the 7595bp vector fragment was recovered;

[0037] 2) Using primer 1 (sequence shown in SEQ ID NO1) and primer 2 (sequence shown in SEQ ID NO2), using pLVX-Large T-EF1-EGFP as a template to amplify the Large T-T2A fragment with a length of 2155bp ; Using primer 3 (sequence shown in SEQ ID NO3) and primer 4 (sequence shown in SEQ ID NO4), using pLVX-sgRNA-mCherry as a template to amplify the T2A-mCherry fragment, the length is 774bp;

[0038] 3) Using the Large T-T2A fragment and the T2A-mCherry fragment obtained in step 2) as templates, adding primer 1 and primer 4, performing overlapping PCR amplification, and obtaining a DNA fragment with a length of 2875 bp of the LargeT-T2A-mCHRRY fragment;

[0039] 4) Recover throu...

Embodiment 2

[0042] A method for constructing a reversible and inducible porcine ovary granulosa cell line, comprising the steps of:

[0043] 1) Isolation and cultivation of granulosa cells:

[0044] Pig ovary samples were obtained from a commercial slaughterhouse, brought back to the laboratory in warm sterile saline (38°C) within 2 h, and then transferred to the laboratory. Use a 10mL syringe to absorb the oocytes and ovarian granulosa cells in the follicular fluid, then remove the oocytes and the complex of oocytes and granulosa cells with a mouth pipette, collect the porcine ovarian granulosa cells, and centrifuge at 600g for 5 minutes to collect the cells and wash 3 times, grow to 10cm 2 In a petri dish, the culture system uses 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;

[0045] 2) Lentivirus packaging and cell infecti...

Embodiment 3

[0050] Example 3: GCs isolation and infection of Large T gene

[0051] The morphology of GCs isolated and cultured in the present invention is short and fibrous (such as figure 2 Shown in A), by pLVX-Tet3G-Large T-T2A-mCherry-Puro r Lentiviral transfection, while adding Dox induction, showed that GCs cell transgenes (such as figure 2 Shown in B and 2C); by adding puromycin for selection, the Large T gene was successfully transfected, and the positive rate of the cells reached 100% to express mCherry, while maintaining the morphology and rapid proliferation of fibroblasts, continuously cultured in vitro for more than 6 months (such as figure 2 Shown in D and 2E), while the non-transgenic primary cells, cultured to the 5th day, the morphology began to change obviously, and the refraction and state became worse (such as figure 2 F shown).

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Abstract

The invention discloses a construction method of a conditioned cell immortalization lentiviral vector. The invention also discloses the pLVX-Tet3G-Large T-T2A-mCherry-Puro<r> lentivirus obtained by packaging with the lentiviral vector and application thereof in establishment of a porcine granulosa cell line. The invention for the first time establishes a reversible and inducible porcine granulosacell line through a Tet-on-3G system, the cell line has inducible and reversible characteristics. When Dox is added, cell proliferation can be stably maintained in vitro; when Dox is removed, granulosa cells can be differentiated into functional terminal cells to secrete more estrogens and progesterones, but loses proliferation ability at the moment; and when Dox is added again, the cells can restore to a proliferation state. The reversible cell line is of important significance for study of cell proliferation, differentiation and dedifferentiation. At the same time, according to the biological properties of granulosa cells, the cell line provided by the invention has important application value for study of granulosa cell hormone secretion and oocyte development regulation.

Description

technical field [0001] The invention relates to the field of genetic bioengineering, in particular to a lentiviral vector and its construction method and its application in porcine ovary granulosa cell lines Background technique [0002] Ovarian granulosa cells (Granulosa cells, GCs) are an important type of cells in follicles, which secrete estrogen and progesterone, and play an important role in regulating oocyte development, ovulation and pregnancy maintenance. The newly isolated GCs cultured in vitro are of great value in the application of research on estrogen synthesis and oocyte development, and at the same time provide a basis for the identification of methods for the diagnosis and treatment of female reproductive diseases, and provide technologies for reducing reproductive losses in livestock. support. However, GCs cultured in vitro are easy to differentiate and have limited proliferation potential, which limits its application and research. [0003] Many research...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/66C12N5/10C12R1/91
CPCC12N5/0682C12N15/86C12N2510/00C12N2740/15043
Inventor 白银山朱翠冯美莹张守全詹小舒李巨浪
Owner FOSHAN UNIVERSITY
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