The Factors Influence On The Establishment Of Embryonic Germ Cells Lines In WuZhiShan Inbreeding Pigs

A total of 200 fetuses were collected from 18 WuZhiShan inbreeding Pigs (WZSP) to study the factors influence on the establishment of porcine embryonic germ (EG) cells lines. The factors included feeder cells, condition medium (CM), growth factors and digesting enzymes. Moreover, EG cells evaluation was carried out.(1) Porcine embryonic fibroblasts (PEF) were obtained from 26²8d fetuses of WZSP, they could be purified by passaging. PEF were passaged to F₁₀, it was shown that PEF could be cultured long-term in vitro and preserved at -196℃. The normal karyotype of PEF was found out at F₅ by karyotype analysis.(2) To study the effect of feeder cells on porcine EG cells cultured in vitro, 53 fetuses were collected from 26.5d pregnant gilts. 33 of them were plated on STO feeder cells and 20 were co-cultured with PEF. When using the same medium, porcine PGC-derived colonies cultured on STO feeder cells could grow well. The colonies co-cultured with PEF could develop in F₀, but they differentiated after passage. So PEF could not maintain the growth of porcine EG cells, while STO feeder cells could do.(3) To study the effect of condition medium (CM) on porcine EG cells cultured in vitro, 35 fetuses were collected from 26.5d pregnant gilts. 15 of them were cultured in BRL-CM and 20 were in common medium (DMEM+15%FCS+2mmol/ml L-glutamine +0.1mmol/ml MEM non-essential amino acids +0.1mmol/ml β - mercaptoethanol +100IU/ml Penicillin+100 μ g/ml Streptomycin). The results manifested that both BRL-CM and common medium were suitable for porcine EG cells culturing and passaging when having STO feeder cells. So BRL-CM can be used to culture porcine EG cells.(4) To study the effect of growth factors on porcine EG cells cultured in vitro, 78 fetuses were collected from 26.5d pregnant gilts. 35 of them were cultured in medium supplemented with LIF, SCF and bFGF (medium â…¡) and 43 were cultured in common medium (medium â… ). The results showed that both medium â…  and medium â…¡ could be used culturing and passaging porcine EG cells when having STO feeder cells, but medium II was better than medium â…  .So porcine EG cells can also be cultured in medium without growth factors.(5) To study the effect of digesting enzymes on passage porcine EG cells, 34 fetuses were collected from 26.5d pregnant gilts. PGCs were isolated and plated on STO feeder cells. On passaging, the colonies were digested by dispase or 0.25% trypsin-EDTA. The result was that renascent colonies of EG cells could be obtained and passaged at both cases. It indicated that both dispase and 0.25% trypsin-EDTA can be used to passage porcine EG cells.(6) The colonies of F₃^F₄ EG cells were identified. The morphology of porcine PGC-derived colonies was multiplicity, cells were tightly packed in colonies and colonies edge was plain. The colonies were AKP-positive, reacted with SSEA-1 antibody and their immunity fluorescence staining of Oct-4 was positive. The colonies placed in suspension culture in medium containing calf serum without growth factors differentiated into simple embryoid bodies (EBs). These cells have many characteristics...