| Chicken primordial germ cells (PGCs) were the ancestor of all level germ cell and transgenic animals have become important tools for biological research. Transgenic chicken could be obtained by transfection the PGCs in vivo or in vitro and screening the offsprings. In the present research, the migration route of PGCs was identified by monoclone antibodies. According to location and content, the PGCs were transfected by the plasmids and the transgenic offsprings were gotten. Meanwhile, a new method, interspecies nuclear transfer and chicken chimera, was explored for the possibility of production transgenic chicken.Stage-specific embryonic antigen-1 (SSEA-1) is a carbohydrate epitope associated with chicken PGCs development, such as PGCs adhesion, migration and differentiation. In the present study, MC-480, monoclonal antibodies against SSEA-1, was employed to identify the migration of PGCs. SSEA-1 positive cells were identified in the germinal crescent. Based on location and morphology, these cells were identified as PGCs. Circulating PGCs through embryonic blood vessels were also SSEA-1 positive. At about the stage 29, PGCs in the dorsal mesentery and gonad could be identified using the monoclonal antibody MC-480. Meanwhile, 2C9 Monoclonal antibody anti-PGCs was also used to recognized PGCs during the migration. The positive cells was in the germinal crescents blood vessel and germinal gonad. The presence of PGCs at corresponding stages was verified using periodic acid Schiff stain.The concentration of PGCs of White Leghorn in male and female were presented during the early developmental stages 13-17. A multiple polymerase chain reaction (M-PCR), which utilized two pairs of primers, was employed to identify the sex of chicken embryos. The concentrations of PGCs per u 1 in the blood of female at stages 13s 14, 15s 16 and 17 were 65.00s 53.65s 14.58s 9.44 and 2.22, respectively. And those in male at the same stages were 19.64s 29.22s 18.08s4.44 and 2.08, respectively. At stages 13 and 14, female chicken embryos have more PGCs than male chicken embryos (p<0.05). In the later stages 15s 16 and 17, however, the concentrations of PGCs in female and male were of no difference (p>0.05). It is concluded that the circulating PGCs concentration in female and male was different at early stages 13-14 but the difference gradually disappeared with the embryo development.When embryos of chicken at stages 14 and 15 of development were microinjected with DMEM culture solution into the blood vessel, the embryos were damaged and lost internal PGCs. An analysis of the number of PGCs in the germinal ridge showed that the damage of microinjection cause decrease of the number of the PGCs. The number of PGCs of damaged female embryo was 80.6% to that of the normal.Two plasmid vectors pEGFP-N1 and pEOVALG cany ing the GFP gene were injected into the aortal dorsal of stages 14-16 chicken embryos and PGCs in the blood were transfected. Subsequently, the eggs were allowed to hatch, and the chickens were screened for the GFP gene by using the polymerase chain reaction. In the group of pEGFP-Nl, four female chicken that survived to sexual maturity contained the GFP gene in their blood. Subsequently, these females were mated with wild-type male chicken. The germline transmission rates are ranging from 5% to 53.6%. The 471bp fragment of generation 1 (G1), the primers was designed by GFP gene in the plasmid pEGFP-Nl, was blast with nuclear acide, and the result showed that the fragment was consist with the GFP gene in the plasmid pEGFP-Nl. The GFP gene was expressed in the Gl chicken whole body cultured for 36 h, and it also espressed in Gl kidney But in the group of pEOVALG, one female and two males survived to sexual maturity contained the GFP gene in their blood, there is no offspring was obtained when the chicken mated with the oppositive sex.Chicken-rabbit interclass cloned embryos reconstructured from blastodermal cells with enucleated metaphase II oocytes can develop to the blastocyst stage. When chicken blastodermal cel... |
PDF Full Text Request