Application of vascular endothelial cell growth factor in promotion of 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 problems such as instability and inefficiency

Active Publication Date: 2021-08-13
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 cell growth factor in promotion of proliferation and migration of chicken primordial germ cells
  • Application of vascular endothelial cell growth factor in promotion of proliferation and migration of chicken primordial germ cells
  • Application of vascular endothelial cell growth factor in promotion of 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 fluid

[0021] (1) VEGF basal culture medium:

[0022] KO-DMEM, 2.5% chicken serum, 7.5% fetal bovine serum, 1.2mM sodium pyruvate (purchased from Beijing Suo Lai Bao Technology Co., Ltd.), 100ug / ml heparin sodium (purchased from Beijing Suo Lai Bao 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 nucleotide supplement (nucleotide Supplement, purchased from Merck Millipore China Co., Ltd.), 0.1mM β-mercaptoethanol, 10ng / ml hFGFb (human basic fibroblast growth factor, purchased from Beijing Suo Lai Bao Technology Co., Ltd.), 1X NEAA (not essential 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 f...

Embodiment 2

[0025] Embodiment 2 chicken primordial germ cell culture and VEGF treatment

[0026] The chicken variety used in the test is Hongyao chicken (produced in Suqian, Jiangsu, and the implementation of the present invention is not limited to this variety). The eggs are hatched to the 14-17HH stage under the condition of 60% (using a commercial chicken embryo incubator) at 37.8 ° C and relative humidity (incubation for 60 hours), disinfect the eggshell with alcohol, place the egg in a petri dish after opening, collect 5-10 μL of blood from the yolk vein, heart, etc. by siphoning with a glass pipette, and add the blood to a medium containing PBS (phosphate buffer saline) , purchased from Thermo Fisher Scientific Co., Ltd.) in a 1.5mL centrifuge tube. Transfer the isolated chicken primordial germ cells into a 24-well cell culture plate, add freshly prepared VEGF basal culture medium, and place at 39°C with 5% CO 2 Cultivate in an incubator for 3 days, change the medium in half every ...

Embodiment 3

[0028] Example 3 Detection of Cell Proliferation

[0029] (1) Chicken primordial germ cells in a 96-well plate were treated with VEGF and cultured for 1d, 3d, and 5d, 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 450nm. See the test results figure 1 . The present invention finds that VEGF of 25 and 50 ng / mL can improve the proliferation rate and survival rate of chicken primordial germ cells when culturing for 1-3 days, wherein VEGF of 25 ng / mL has a very significant effect on promoting proliferation, and VEGF of 50 ng / mL can be used for 5 days. Chicken primordial germ cell proliferation rate and survival rate will play a certain inhibitory effect, so this embodiment has determined 25ng / mL VEGF as the optimal treatment concentration of the culture system of this embodiment, with cultivating 3d as the optimal concentration of this...

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Abstract

The invention belongs to the technical field of poultry breeding and reproduction management, and particularly relates to application of a vascular endothelial cell growth factor in promoting proliferation and migration of chicken primordial germ cells. The influence of vascular endothelial cell growth factors (VEGF) on the proliferation and migration capabilities of chicken primordial germ cells is compared. VEGF with a proper concentration is added on the basis of an existing chicken primordial germ cell culture solution, proliferation of chicken primordial germ cells can be promoted, the added VEGF with different concentrations has different promotion effects on proliferation of the chicken primordial germ cells, and the application effect is optimal when the concentration of the VEGF in a blood vessel is 25ng / mL and treatment is carried out for 3 days. The method can effectively promote the proliferation of the chicken primordial germ cells in vitro, the VEGF with proper concentration in the basic culture solution can obviously improve the capability of migrating and planting the chicken primordial germ cells to gonads, and the biological characteristics are kept 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 of migrating and colonizing gonads. Background technique [0002] Primordial germ cells (PGCs) are the 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 reinject embryonic blood vessels to migrate to the gonads 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...

Claims

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Application Information

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Patent Type & Authority Applications(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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