Female chicken primordial germ cell expansion in vitro culture system and application thereof
A technology of primordial germ cells and culture system, which is applied in the field of in vitro expansion and culture system of primordial germ cells of female chickens, and can solve problems such as inapplicability
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Embodiment 1
[0031] (1) Preparation of aromatase culture medium
[0032] Find the sequence of the aromatase gene CYP19A1 (ENSGALG00000013294) on the ENSEMBL website, design primers (upstream: 5'-AGAAAACCTACTCAGA-3', downstream: 5'-GGCTTACAATAATGGTAG-3'), and use the gonad cDNA as a template to amplify the gene fragment. Use the In-Fusion kit (In-Fusion HD Cloning Kit, TaKaRa Company) to construct a vector with the target DNA, transform Escherichia coli DH5α (TaKaRa Company), pick positive clones, and send them to be sequenced to determine the sequence and expand the culture. Toxin plasmid extraction kit (Endo-free Plasmid mimi Kit II, OMEGA) was used to extract positive clone plasmid p-EGFP-CYP. Recovery of DF1 cells ( CRL-12203 TM , ATCC company) to 10cm cell culture dish (Thermo company), 37 ℃, 5% CO 2 Culture under conditions (the culture system is KO-DMEM (Gibco Company) with a volume ratio of 90%, fetal bovine serum (Gibco Company) with a volume ratio of 10%, and when the cells g...
Embodiment 2
[0041] The eggs of Huiyang bearded chickens (from the original breeding farm of the Institute of Animal Science, Guangdong Academy of Agricultural Sciences) were incubated for 5 to 7 days, and the chicken primordial germ cells were isolated from the gonad tissue of the chicken, and DNA was extracted from the limbs for sex identification. After sex identification, the female chicken primordial germ cells were inoculated onto the culture system and the rat liver cell feeder layer treated with the radioactive source in step (3) of Example 1, at 37° C., 5% CO 2 Conditioned incubator culture. The culture system is: 52% (v / v) KO-DMEM, 30% (v / v) rat liver cell culture medium, 7.5% (v / v) fetal bovine serum, 2.5% (v / v) chicken serum, 1% (v / v) non-essential amino acids, 1% (v / v) GlutaMAXTM, 1% (v / v) pyruvate, 1% (v / v) β-mercaptoethanol, 1% (v / v) penicillin Streptomycin, 3% (v / v) aromatase broth, 4ng / mL rmSCF, 2.5ng / mL rhFGF. A total of 16 female chicken embryos were isolated this time...
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