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Application of 25-hydroxycholesterol in promoting the proliferation of chicken primordial germ cells

A technology of primordial germ cells and hydroxycholesterol, which is applied in the application field of 25-hydroxycholesterol in promoting the proliferation of chicken primordial germ cells, and can solve problems such as researches that have not been reported yet

Active Publication Date: 2017-12-01
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that the Sonic Hedgehog signaling pathway is conserved in chickens, but the research on the Sonic Hedgehog signaling pathway in chicken germ cells has not been reported

Method used

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  • Application of 25-hydroxycholesterol in promoting the proliferation of chicken primordial germ cells
  • Application of 25-hydroxycholesterol in promoting the proliferation of chicken primordial germ cells
  • Application of 25-hydroxycholesterol in promoting the proliferation of chicken primordial germ cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] 1. Preparation of culture medium

[0015] (1) cKO medium

[0016] KO-DMEM 75.8%, KO-BRl-CM 10%, Hyclone Defined FBS 7.5%, Chicken serum2.5%, GS nucleoside supplement 1x, pen / strep 1x, GlutaMAX 1x, b-Mercaptoethanol0.1mM, hFGFb 4ng / ml

[0017] (2) 25-hydroxycholesterol culture medium

[0018] KO-DMEM 75.8%, KO-BRl-CM 10%, Hyclone Defined FBS 7.5%, Chicken serum2.5%, GS nucleoside supplement 1x, pen / strep 1x, GlutaMAX 1x, b-Mercaptoethanol0.1mM, hFGFb 4ng / ml, 25-Hydroxycholesterol (25-hydroxycholesterol) 0.1 μM, 0.5 μM, 1 μM.

[0019] 2. Inoculation of cells

[0020] (1) Thaw PGC cells and inoculate them into a 35mm culture dish covered with a feeder layer, and subculture when they are overgrown.

[0021] (2) Cell count

[0022] 3. Add 25-hydroxycholesterol

[0023] After cell counting, the culture medium was divided into 4 tubes, each with 6 × 10 4 1 PGC, centrifuged, sucked away the supernatant, then resuspended with cKO culture medium (control group), cKO cultur...

Embodiment 2

[0030] Use cKO medium and cKO medium containing 1 μM 25-hydroxycholesterol to culture PGCs for three days respectively, use OMEGA RNA extraction kit (CAT#R6834-01) and Tiangen reverse transcription kit (CAT#KR106- 02), respectively obtained the cDNA of PGC after three days of culture in three kinds of culture medium, then utilized the NCBI database (http: / / www.ncbi.nlm.nih.gov) to search for CVH, CDH, DAZL, Nanog, PouV sequences, and designed primers ( The primer sequence is shown in Table 2), with the obtained cDNA as a template, carry out RT-PCR with corresponding primers, and the PCR product is identified by 2% agarose gel electrophoresis, see figure 2 , the results showed that CVH, CDH, DAZL, Nanog, PouV, these 5 genes were still highly expressed after being cultured in the medium containing 1 μM 25-hydroxycholesterol, indicating that this medium can well maintain chicken primordial germ cells biological characteristics.

[0031] Table 2 RT-PCR primer sequences

[0032]...

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Abstract

The invention discloses the application of 25-hydroxycholesterol in promoting the proliferation of chicken primordial germ cells. The inventor has studied in depth the impact of 25-hydroxycholesterol on the proliferation of chicken primordial germ cells. The results showed that adding 25‑hydroxycholesterol to the existing culture medium of chicken primordial germ cells could promote the proliferation of chicken primordial germ cells. According to the difference of adding 25-hydroxycholesterol concentration, its promotion degree to chicken primordial germ cell proliferation is different, and when 25-hydroxycholesterol concentration is 1 μ M, its action effect is the most remarkable, can effectively promote chicken primordial germ cell proliferation in vitro Proliferate, and chicken primordial germ cells can well maintain their biological characteristics in the culture medium containing this concentration of 25-hydroxycholesterol.

Description

technical field [0001] The invention belongs to the application field of 25-hydroxycholesterol, in particular to the application of 25-hydroxycholesterol in promoting the proliferation of chicken primordial germ cells. Background technique [0002] Chicken primordial germ cells (PGCs), as the precursor cells to produce sperm and eggs, can pass genes from the previous generation to the next generation, and have broad application prospects in the production of transgenic chickens and the preservation of genetic resources of endangered species. On the one hand, transgenic chickens can be used in breeding work to produce high-quality breeds with disease resistance and high yield; on the other hand, they can be used as bioreactors to produce bioactive substances such as medical proteins. It is of great significance to use cryopreserved chicken PGCs to preserve the quality resources of high-quality breeds, especially endangered breeds. The establishment of an in vitro culture sys...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/075C12N5/076
Inventor 陆阳清陈东阳魏巍张文馨陈美娟
Owner GUANGXI UNIV
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