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Mouse ovary primary cell culture medium and in-vitro culture method capable of being applied to gene editing

A primary cell, in vitro culture technology, applied in the field of cell culture, can solve the problem of lack of in vitro culture method of mouse primary cells, achieve high gene editing efficiency, promote growth and survival, and achieve good results

Pending Publication Date: 2021-10-12
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is: the lack of a method for culturing primary mouse cells in vitro that can be used for efficient gene editing

Method used

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  • Mouse ovary primary cell culture medium and in-vitro culture method capable of being applied to gene editing
  • Mouse ovary primary cell culture medium and in-vitro culture method capable of being applied to gene editing
  • Mouse ovary primary cell culture medium and in-vitro culture method capable of being applied to gene editing

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preparation example Construction

[0039] The present invention also provides a kind of preparation method of mouse ovary primary cell culture medium, comprises the following steps: prepare 50ml of DMEM / F-12 (1:1) complete culture medium in ice bath, wherein add BSA, control concentration is 100-300μg / ml; add L-ascorbic acid, control the concentration at 20-40μg / ml; add insulin, control the concentration at 0.25-2g / L; add transferrin, control the concentration at 0.55-1.1g / L; add selenium sodium nitrate, the control concentration is 0.0003~0.001g / L; add fetal bovine serum, control concentration is 10%~20%; add epidermal growth factor EGF, control concentration is 20~100ng / ml; add recombinant protein RSPO1, control concentration 150-500ng / ml; adding fibroblast growth factor 10, the control concentration is 5-30ng / ml; adding N-acetyl-L-cysteine, the control concentration is 0.5-2mM; adding nicotinamide, the control concentration is 5~20mmol / L; add human Wnt-3a protein, control concentration is 10~50ng / ml; add pen...

Embodiment 1

[0049] Example 1 Preparation of mouse ovary primary cell culture medium

[0050] Prepare 50ml of DMEM / F-12 (1:1) complete medium in an ice bath, in which the concentration of BSA is 150μg / ml, the concentration of L-ascorbic acid is 30μg / ml, the concentration of insulin is 1g / L, and the concentration of transferrin The concentration of sodium selenite is 0.55g / L, the concentration of sodium selenite is 0.0065g / L, the concentration of fetal bovine serum (10%-20%), the concentration of epidermal growth factor EGF is 50ng / ml, the concentration of recombinant protein RSPO1 is 250ng / ml, and the concentration of fibroblast Cell growth factor 10 concentration is 15ng / ml, N-acetyl-L-cysteine ​​concentration is 1mM, nicotinamide 10mmol / L, human Wnt-3a protein concentration is 20ng / ml, penicillin 75U / ml, streptomycin 80 μg / ml, adenylyl cyclase activator 2 μmol / L, mix well under ice bath conditions, and store in 4°C refrigerator.

Embodiment 2

[0051] Example 2 Isolation and primary culture of primary cells after digestion of mouse ovary tissue

[0052] The specific operation steps are as follows:

[0053] (1) Disinfect and sterilize the ultra-clean bench and experimental equipment.

[0054] (2) Select 2 C57 female mice aged 4-8 weeks (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) and put them to death by cervical dislocation, soak the mouse body surface with 75% alcohol for 2 minutes; absorb water The paper absorbs the excess alcohol outside the mouse body surface.

[0055] (3) In the ultra-clean bench, place the mice on a clean operating board. Lift the abdominal skin of the mouse with tweezers in one hand, and cut the abdominal skin of the mouse with the other hand to expose the abdominal cavity of the mouse.

[0056] (4) Find the uterus on the left and right sides of the mouse, and remove the ovary.

[0057] (5) Place the obtained ovarian tissue in a pre-cooled sterile PBS solution containin...

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Abstract

The invention belongs to the technical field of cell culture and particularly relates to a mouse ovary primary cell culture medium and in-vitro culture method capable of being applied to gene editing. In order to solve the problem that a mouse primary cell in-vitro culture method capable of being applied to efficient gene editing is lacked at present, the mouse ovary primary cell culture medium capable of being applied to gene editing is provided and comprises BSA storage liquid, L-ascorbic acid, insulin, transferrin, sodium selenite, fetal calf serum, an epidermal growth factor (EGF), a recombinant protein RSPO1, a fibroblast growth factor 10, N-acetyl-L-cysteine, nicotinamide, a human Wnt-3a protein, penicillin and streptomycin double antibodies and a DMEM / F-12 (1: 1) complete culture medium. When the culture medium provided by the invention is used for culturing ovarian primary cells, the cell survival rate is high, the obtained cells can be applied to gene editing, the efficiency reaches up to 90%, and the application prospect is wide.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to a mouse ovary primary cell culture medium and an in vitro culture method applicable to gene editing. Background technique [0002] The ovary is the reproductive organ of female animals. Its main function is to produce eggs and secrete steroid hormones. The ovarian epithelium and stroma are composed of granulosa cells, oocytes, theca cells, interstitial cells and other cells. The cells interact to coordinately regulate the synthesis of steroid hormones in female animals, maintain the survival, growth, development and maturation of cells, and ensure the normal physiological functions of the ovary. The primary cell culture technology of ovarian granulosa cells, oocytes, follicular cells, mesenchymal cells, etc. is an important method for the study of reproduction. [0003] At present, there are few studies on the culture technology of ovarian primary cells. Patent...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/075
CPCC12N5/0609C12N2500/38C12N2500/25C12N2501/11C12N2501/119C12N2500/32C12N2501/415C12N2501/998
Inventor 任柯星
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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