Early Development Rules Of Chicken Embryo And The Related Transgenic Technology

With the rapid development of embryonic and genetic engineering in chick, the reseach in the embryonic development biology demands a uniform criterion for the embryonic development rules in chick, and the reseach on the early-stage embryonic development rules in vivo or in vitro in chick is necessary for fowl breeding and transgenic research. Through the investigation on the early-stage and incubating-stage embryonic development and organ growth in chick, the study explored the growth rules of chick embryo. Moreover, through direct microinjection of human growth hormone(hgh) gene and human tissue kallikein-1(KLK1) gene into the germinal disc of developing embryos in chick, the study explored the new approaches for producing transgenic chickens.1,The early embryo development rule in chicken: By slaughtering the chick on different time periods after laying an egg, the developing fertilized eggs were obtained from the oviduct or uterine to be observed and investigated under the microscope. The results showed that: the first cleavage happened 5 hours(0-0.5 hour of uterine age)after laying an egg, morular stage happened 11.5 hours(6-6.5 hours of uterine age)after laying an egg, blastocyst stage happened 15.5 hours(10-10.5 hours of uterine age)after laying an egg. According to morphological condition of the blastodisc, the embryo development can be divided into two stages: cleavage stage, from 5.5 hours(0-1 hour of uterine age) to 15.5 hours(10-10.5 hours of uterine age) after laying an egg, was characteristic of the formation of a blastodisc by 6-7 layers of cells; and blastocyst stage, from 17.5 hours(12-12.5 hours of uterine age) after laying an egg to pellucida area forming period, was characteristic of the formation of a light area and a dark area at the center of the blastodisc.2,ES were isolated from the stage X of chicken embryos with 0.25% trypsin-0.04% EDTA, then cultured in the culture system 4 with high glucose DMEM supplemented with 10% fetal bovine serum, 2% chicken serum, 2mmol/L L-glutamine, 5.5×10-5β-mercaptoethanol, 1mmol/L sodium pyruvatel, with leukemia inhibitory factor(LIF, 1000U/ml), basic fibroblast growth factor(bFGF, 10ng/ml) and stem cell factor(SCF, 5ng/ml), ES-like colonies were developed 2-5 days later, and there were 1 to 5 passages of ES-derived AKP positive colonies formed, with the values of 80%, 66.67%, 46.67%, 26.67% and 3.33% respectively. However, there was no AKP positive colony observed in culture system 1, the formation of one passage ES-derived AKP positive colonies was 33.33% in this system 2, and in system 3, there were 1st and 2nd passages of ES AKP positive colonies observed, with the value of 40% and 20%, respectively. The 1st to 5th passage of ES were characterized by morphology, colony formation, proliferation capability, karyotype, alkaline phosphatase activity and cell differentiation. Results showed that EScultured in the culture system 4 could be subcultured for five times and still maintained undifferentiated situation with normal chromosome karyotype. It seemed that the cultured chicken ES maintained characters of pluripotent stem cells.3,Transfection of exogenous genes into the germinal disc of chicken: Through direct microinjection of human growth hormone(hgh) gene and human tissue kallikein-1(KLK1) gene into the germinal disc of 320 developing chicken embryos of 24 hours after incubating, 18 chicks(5.6%) were hatched, but 8 died respectively 1, 2, 3 and 5 days after hatch, and 7 died 3 months later, only 3 survived. The dead chicks showed some symptoms like standing unsteadily and wryneck., while the survived exhibited no abnormalties. The genomic DNA were extracted from the tissues of 150 dead embryos and 18 chicks and conducted DNA hybridization test to detect the integration of exogenous genes. The results showed that: 3 embryo samples and 3 chick samples were detected hgh-positive, the integration rate was 12.5%; 18 embryo samples and 6 chick samples were detected KLK1-positive, the integration rate was 22.2%. The findings demonstrated that direct microinjection of foreign genes into the germinal disc of developing embryos in chick was an effective method to make transgenic chicken.