Study On Embryonic Germ Cells Chimera Potential Of Guanzhong Diary Goat

Both embryonic stem (ES) cells and embryonic germ (EG) cells maintained the features of self-renewal and pluripotency. One test is conducted by injecting ES or EG cells from a donor animal into the cavity of recipient blastocysts. The combination embryos were then transferred to the uterus of a pseudo-pregnant female animal treated in synchronization of estrus. The progeny resulting from hybrid blastocysts was a chimera. The major object of this paper is to explore whether chimera can be generated by GuangZhong milk goat embryonic germ cells or not. Results of this study provide information to understand chimeral animals which may be used in the future for clinical application. The results obtained were as follows:1. EG cells isolated from white Guanzhong goat were cultured for 3-4 passages and incubated in 0.05% trypsin, 0.02 % EDTA for 30 min. Cells formed small clusters containing 5-15 cells each was delivered into the blastocoele cavity that was isolated from a nature mated black goat By using the microinjector. 29 hybrid blastocysts were incubated in a CO2 incubator for 30 min and then immediately transferred to 9 recipient goats that had been treated by estrus synchronization. One of the implant goats (NO. 46) was pregnant day147 which was transferred 2 hybrid blastocysts, but gave birth to two lambs and a dead malformed fetus. The fetus gender was undetermined. One new born lamb was a black male and the other one was black/white female that had white stripe hairs in front of head. The malformed fetus also had more white hairs in the area of the head than female lamb.2. Ten microsatellite markers including BMS1004, BMS1290, BM203, BMS875, BMS574, SR-CRSP1, SR-CRSP5, SR-CRSP24, OarAE101 and OarFCB11 were selected to characterize the chimerism of newborn lambs. The results showed that female lamb and malformed fetus had the identical DNA pattern with EG cells pattern which proved that EG cells were chimera into female lamb and malformed fetus. There were ten markers that demonstrated female lamb and malformed fetus having same pattern, but markers BMS875, BMS574 and BMS1004 that showed significantly different with male lamb. This result revealed that female lamb and malformed fetus were chimera lamb and monozygotic twins. 3. To further characterize the chimera goat, To determine if the newborn lambs were chimeras, Sry gene was analyzed by PCR assay. Aml-x gene (X amelogenin) co-amplified in PCR assay was used as internal control to detect the x-chromosome marker. If the hybrid blastocysts developed to form a fetus, the chimera should preserve two features, white hairs and Sry gene positive, especially in a female animal. The result showed that female lamb and malformed fetus not only carried black/white hairs in front of its head, but also maintained the Sry gene although it was a female goat indicating that both animals were chimera goats.