Application of vascular endothelial growth factor in promoting the proliferation and migration of chicken primordial germ cells

A technology of primordial germ cells and growth factors, applied in the direction of germ cells, animal cells, embryonic cells, etc., can solve the problems of low efficiency and instability

Active Publication Date: 2022-07-29
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods are still unstable and inefficient

Method used

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  • Application of vascular endothelial growth factor in promoting the proliferation and migration of chicken primordial germ cells
  • Application of vascular endothelial growth factor in promoting the proliferation and migration of chicken primordial germ cells
  • Application of vascular endothelial growth factor in promoting the proliferation and migration of chicken primordial germ cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] The preparation of embodiment 1 culture liquid

[0021] (1) VEGF basal medium:

[0022] KO-DMEM, 2.5% chicken serum, 7.5% fetal bovine serum, 1.2mM sodium pyruvate (purchased from Beijing Soleibo Technology Co., Ltd.), 100ug / ml heparin sodium (purchased from Beijing Soleibo Technology Co., Ltd.), 1X antibiotic-antimycotic (antibacterial-antifungal agent, purchased from Thermo Fisher Scientific Co., Ltd.), 1X GlutaMax (glutamine additive, purchased from Thermo Fisher Scientific Co., Ltd.), 1X GS nucleoside supplement (nucleotides) Supplement, purchased from Merck Millipore China Co., Ltd.), 0.1mM β-mercaptoethanol, 10ng / ml hFGFb (human basic fibroblast growth factor, purchased from Beijing Soleibo Technology Co., Ltd.), 1X NEAA (optional) Amino acid solution, purchased from Thermo Fisher Scientific Co., Ltd.), 1XB-27supplement (B-27 supplement, purchased from Thermo Fisher Scientific Co., Ltd.), 25ng / mL Human Activin A (Human Activin A, purchased from From Paiptech Bio...

Embodiment 2

[0025] Example 2 Chicken primordial germ cell culture and VEGF treatment

[0026] The chicken breed used in the test is Hongyao chicken (produced in Suqian, Jiangsu, the implementation of the present invention is not limited to this breed), and the eggs were incubated at 37.8° C. and the relative humidity of 60% (using a commercial chicken embryo incubator) to the 14-17HH stage. (60h incubation), sterilize the egg shell with alcohol, put the egg in a petri dish after opening, collect 5-10 μL of blood from the yolk vein, heart, etc. with a glass pipette by siphoning, add the blood to PBS (phosphate buffered saline) , purchased from Thermo Fisher Scientific Co., Ltd.) in a 1.5 mL centrifuge tube. Transfer the isolated chicken primordial germ cells into a 24-well cell culture plate, add freshly prepared VEGF basal medium, and place at 39°C with 5% CO. 2 Cultured in the incubator for 3 days, half of the medium was changed every other day, and subculture could be carried out after...

Embodiment 3

[0028] Example 3 Cell Proliferation Detection

[0029] (1) Chicken primordial germ cells in a 96-well plate were cultured for 1 d, 3 d and 5 d after adding VEGF treatment, and then 10 μL of CCK-8 mixture (purchased from Beijing Boaosen Biotechnology Co., Ltd.) was added and incubated for 2 hours.

[0030] (2) Detect the optical density value of the supernatant at 450 nm. See the test results figure 1 . The present invention finds that 25 and 50 ng / mL VEGF treatment can improve the proliferation rate and survival rate of chicken primordial germ cells during 1-3 days of culture, among which 25 ng / mL VEGF has a very significant effect on promoting proliferation, while 50 ng / mL VEGF treatment for 5 days It will have a certain inhibitory effect on the proliferation rate and survival rate of chicken primordial germ cells. Therefore, in this example, 25ng / mL of VEGF was determined as the optimal treatment concentration of the culture system of this example, and the culture system w...

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Abstract

The invention belongs to the technical field of poultry breeding and reproduction management, in particular to the application of vascular endothelial cell growth factor in promoting the proliferation and migration of chicken primordial germ cells. The effects of vascular endothelial growth factor (VEGF) on the proliferation and migration of chicken primordial germ cells were compared. Adding an appropriate concentration of VEGF to the existing chicken primordial germ cell culture medium can promote the proliferation of chicken primordial germ cells. Different concentrations of VEGF have different effects on the proliferation of chicken primordial germ cells. When the intravascular VEGF concentration is 25ng / Best results are applied when applied in mL and 3 days of treatment. The invention can effectively promote the proliferation of chicken primordial germ cells in vitro, and a suitable concentration of VEGF in the basal culture medium can significantly improve the ability of chicken primordial germ cells to migrate and colonize gonads, and the biological characteristics remain unchanged.

Description

technical field [0001] The invention belongs to the technical field of poultry breeding and reproductive management. Specifically, it relates to the application field of Vascular Endothelial Growth Factor (VEGF) in promoting the proliferation of chicken primordial germ cells and improving the ability to migrate and colonize gonads. Background technique [0002] Primordial germ cells (PGCs) are progenitor cells that produce sperm and oocytes and can transmit genetic information from parent to offspring. At present, the most efficient and feasible way to produce transgenic chickens is to modify and edit chicken primordial germ cells in vitro, and then re-inject the embryonic blood vessels to migrate to the gonad to develop into gametes (Arjun Challagulla et al 2021. Marker counter-selection via CRISPR). / Cas9 co-targeting for efficient generation of genome edited avian cell lines and germcells. Anim Biotechnol. Mar 2:1-11.). In addition, due to the particularity of chicken b...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0735
CPCC12N5/0611C12N2501/165
Inventor 朱桂玉孟璐满秋黄晓晨侯博文彭哲伦尹彬旭王恒
Owner HUAZHONG AGRI UNIV
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