Differentiation Of Chicken Embryo Primordial Germ Cells In Vitro And Production Of Chimera

Primordial germ cells (PGCs) are the embryonic precursors of the germ cells (ovum and spermatozoa) and adult gametes. PGCs as the pluripotency cells, they have the capability of differentiation similar to embryonic stem cells, they can be cultured and generated in vitro and maintained in undifferentiated states, also have the ability to form chimera. We derived PGCs in chicken embryos at specific developmental stages including the genital ridge at stage 19 embryos and the gonad at stage 28 embryos, and PGCs were isolated by EDTA-Trysin collection method. After cultured and subcultured in vitro, the third passage PGCs were differentiated under the different induced media to get the optimization conditions of differentiation. The other aim of this study was to investigate The possibility of producing chimeras by the transfer of donor PGCs into recipient embryos. The results of this study provide valuble reference for genomic study and medicine invention. The main results were as follows:1. PGCs were induced into osteoblasts under the desamethason, glycerol 2-phosphate disodium salt hydrate and vitamin C. The induced media of different concentrated groupsⅠ,Ⅱ,Ⅲ,Ⅳwere compared, and the differentiated rate was 47%～79%. The inductionⅠandⅡshowed the best result, and there was no significant difference between them (P>0.05), but they were remarkably better than the other two mediaⅢandⅣ(P<0.01). There was no significant difference between stage 19 and stage 28 under the same medium(P>0.05). After differentiating for 21days, the results showed positive for ALP, the Von Kossa's stain and the immunohistochemistry stain respectively, which suggesting that PGCs could be differentiated into osteoblasts under different conditions in vitro.2. PGCs were induced into adipocytes under the desamethason, insulin and IBMX. The induced media of different concentrated groupsⅠ,Ⅱ,Ⅲ,Ⅳ,Ⅴwere compared, and the differentiated rate was 75%~89%. Compared with single use of inductionⅠandⅡ, the inductionⅠc ombined withⅤshowed the best results(P<0.01). There was no significant difference between the inductionⅠandⅡ(P>0.05), but compared with the inductionⅡ, the adipocytes appeared earlier under the inductionⅠ. The inductionⅢ andⅣshowed no significant difference, and the difference with the other four groups was very remarkable(P<0.01).The rate of the inductionⅤwas only 1%～2%. In the same induction medium, there was no significant difference between stage 19 and stage 28(P>0.05).After induced for 21d, the cells appeared red color with the oil red-O method. Total RNA was abstracted from the differentiated cells, target DNA fragment of 301bp was identied by RT-PCR, which suggesting expression of PPAR-γin the differentiated cells. The results confirmed the capability of PGCs to differentiate into adipocytes.3. PGCs were induced into neuron-like cells under the RA and IBMX. The induced media of different concentrated groupsⅠ,Ⅱ,Ⅲwere compared, the differentiated rate was 75%~87%. InductionⅠa ndⅡwere the best two methods, and there was no significant difference between them(P>0.05), but there was significant difference (P<0.01) compared to inductionⅢ. The results showed that RA and IBMX used singly showed better results than they used together. There was no significant difference between stage 19 and stage 28 under the same medium. Special Nissl body was found by by toluidine blue stain after induced for 4d. Immunohistochemistry results indicated that differentiated cells expressed NSE and NF positive, and GFAP negative. ALL the results showed that PGCs could be differentiated into neuron-like cells.4. The possibility of producing chimeras by the transfer of PGCs into recipient embryos was also investigated in this study. Black-feathery silky fowls and Suqin yellow chickens were used as donors and recipients, respectively. The PGCs of donor are microinjected into the subgerminal cavity of the 40 recipient embryo. Only 2 chickens were hatched to 18d, but there was no phenotypic chimera(feather color chimera); 3 chickens were hatched to 15d, they showed feather color chimeras, the chimeric rate was 7.5%; There were 6～8 chickens hatched to 5～10 d. The positions and areas of the 3 chimeric embryonic black feather were different. Some of the black feather showed in the head, and some showed in the back. The results suggesting that chimera can be got by microinjected PGCs. But no chicken had survived to be born in our experiment, so further research on the methods of production of the chimeric chicken should be exploited.