| In this study,X period blastoderm cells of chicken were studied and isolated to carry on blastoderm embryo culture and scattered culture.The relationship between cell proliferation and differentiation under the two cultures were compared.The model of transgenic chimera chicken was initially established by using electroporation and microinjection technique and X period blastodermal cell for donor cells.This provides a reference for the production of transgenic chicken. The results' are as follows:1.Chicken blastodermal cells at X stage were separately whole embryo cultured and scattering cultured. Observing the condition of cell growth and calculating positive cloning efficiency by dying the cells with AKP.The results showed that there is mostly significant difference in the positive clone rates of subculture between dispersion cells and whole ES cells (p<0.01). The positive rates of the first clone were 73.50%, 57.65% respectively. The positive clone rate of subculture in dispersion cells was significantly different from that of whole ES cells (p<0.01). The positive rates of the second clone were 66.70%,39.80% respectively.2. EGFP was used as the report gene and transfected the second ES cell of chicken by electroporation.Comparing the transfection efficiency under different factors.The factors include:voltage,pulse time,concentration of plasmid, temperature of incubation before and after lightning stroke,cell density,periods of cell growth.The results are as follows:(1)When voltage were setted up as 180 V,230 V,280 V,330 V and 380 V, transfection efficiency were separately 3.01%,5.81%,10.83%,6.70% and 4.08%,transfection efficiency on 280 V was highest and had extreme significant difference compared to other groups(p<0.01).(2)When pulse time were 55μs,75μs,95μs and 115μs, transfection efficiency were separately 10.83％,14.76%,5.69% and 4.09%,the transfection efficiency on 75μs was highest and had extreme significant difference compared to groups of time constant(p<0.01).(3)When concentration of plasmid were 0μg·mL-1,10μg·mL-1,15μg·mL-1,20μg·mL-1 and 25μg·mL-1, transfection efficiency were separately 0%,14.76%,15.91%,18.02% and 12.34%. When concentration of plasmid were from 0μg·mL-1 to 15μg·mL-1,there was on significant difference of survival rate(p>0.05).Transfection efficiency rised with the increase of concentration of plasmid and had significant difference (p<0.05).When concentration were from 20μg·mL-1 to 25μg·mL-1, survival rate and transfection efficiency all began to descend.There were both extreme significant difference on survival rate and transfection efficiency(p<0.01).(4)When temperature of incubation before lighting stroke were 4℃,25℃and 37℃, transfection efficiency were separately 21.52%,18.02% and 12.86%.When temperature was 4℃,there was the highest transfection efficiency. There was no significant difference among the cells under standing 10 minutes of the three temperatures(p>0.05),but there were extreme significant difference among the three temperatures separately(p<0.01).(5)When temperature of incubation after lighting stroke were 4℃,25℃and 37℃, transfection efficiency were separately 21.52%,21.98% and 9.80%. There was no significant difference on survival rate and transfection efficiency between the cells under standing 10 minutes of 4℃and 25℃(p>0.05),but there was significant difference between the two temperatures and 37℃(p<0.01).(6)When cell density were 1×104个·mL-1,1×105个·mL-1,1×106个·mL-1 and 1×107个·mL-1, transfection efficiency were separately 1.89%,21.79%,21.98% and 8.73%.There was effective transfection only when cell density is bigger 1×105个·mL-1.There was no significant difference on survival rate and transfection efficiency when cell density were 1×105个·mL-1 and 1×106 个mL-1(p>0.05).3.There were three methods about windowing on hatching egg before producing the model of chicken chimera,changing shell, windowing on blunt end and windowing on equatorial plane.The hatching efficiency of changing shell was 0.and separately 25% and 31.67% of windowing on blunt end and windowing on equatorial plane.The results of different treatments for hatchability showed that:persistence of normal hatching, windowing without being injected, windowing with being injected, windowing with being injected and transfected were separately 96.7%,5%,14.29% and 5.21%.There was extreme significant difference among them.There were two chicken happened engomphosis on color pattern in the dead embryos collected.One black down feather located in back and one in head and trail.Five chimera chicken which were hatched did not happen conspicuous engomphosis in color pattern.By using PCR to test the DNA picked up from the five surviving chimera chicken,there was no chimerism in the blood. Dissecting and extracting DNA from various kinds of organizations of the five surviving chimera chicken,there were some parts of organizations expressed EGFP gene in four chimera chicken. No.1 was artificial midwifery.At 38th days,it died and showed astasia, legs bent,abnormal appetite and other abnormal phenomena.The PCR amplification showed positive in liver, leg muscle, chest muscle,heart, stomachus glandularis, kidney and genitical gland. No.2 was healthy when it borned but had similar symptoms with NO.1 at 33th day,such as astasia, abnormal appetite, depressed and dired toward evening at 47th day. The PCR amplification showed positive in leg muscle, chest muscle,heart,brain and kidney.No.3 was normal when it born and dired at 23th day.There was no conspicuous abnormal symptom when it dired.There was also no conspicuous symptom of various kinds of organizations and organs after being dissected. The PCR amplification showed positive in liver,lung and stomach. No.4 was a hen and had good condition of growth.It was dissected at 88th and extracted DNA from various kinds of organizations.The production of PCR were all negative.So,there was no engomphosis happened in it. No.5 was a cock and had good condition of growth. It was dissected at 88th and extracted DNA from various kinds of organizations. The PCR amplification showed positive in liver,chest muscle,stomach, kidney and genitical gland.The PCR production of every kind of organizations DNA extracted from chicken of negative control were all negative.
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