| Chicken embryonic primordial germ cells (EPGCs) are the embryonic precursors of the chicken germ cells (ovum and spermatozoa) and adult gametes. Under the co-effect of feeder cell layer and some growth factors, it could develop proliferation and retain undifferentiated. This paper compared the transfection efficiencies of two different methods with EPGCs from chicken embryo at the 19th stage as experiment material, and EGFP as report gene, and emphasized the optimization of electroporation parameters. This paper also focused the concentration of G418 in fast selection through cell toxicity sensitivity experiment, carried out fast selection the transfected EPGCs in vitro, and tried to use it in making transgene chicken by microinjection in the aim of providing some references in producing transgenic chickens. The results showed as following:1. The second generation EPGCs in 19th stage were transfected by calcium phosphate coprecipitation method, liposome-mediated method, electroporation method and calcium phosphate coprecipitation with glycerol shock method with EGFP as report gene. The results indicated that the efficiencies of different method differed, the highest one was 17.096% by electroporation method, then it was 12.554% by calcium phosphate coprecipitation method, followed by calcium phosphate coprecipitation method which was 7.866%, and the lowest one was 1.138% by liposome-mediated method. 2. With EGFP as report gene, the second generation EPGCs of stage 19 was transfected through electroporation in order to find the optimize condition combination including pulse duration, plasmid, concentration, cell density and temperature. The results were as follows: (1) under the same conditions of pulse duration, plasmid concentration, cell density, and temperature, the transfection rates were 12.520%,14.408%,18.635%,17.944% and 13.765% at voltage of 180V,230V,280V,330V and 380V, respectively. The differences between the highest transfection rate at 280V and others expect for that at 330 V were extremely significant (P<0.01); (2) under the same conditions of voltage, plasmid concentration, cell density, and temperature, the transfection rates were 12.520%, 6.143%, 14.715% and 12.368% at pulse duration of 40μs,60μs,80μs and 100μs, respectively. The differences between the highest transfection rate at 60μs and others were extremely significant (P<0.01); (3) under the same conditions of pulse duration, voltage, cell density, and temperature, the transfection rates were 8.300%,12.520%,17.948% and 17.577% at concentration of 5μg·mL-1,10μg·mL-1,15μg·mL-1 and 20μg·mL-1, respectively. The differences between the highest transfection rate at 15μg·mL-1 and others expect for that at 20μg·mL-1 were extremely significant (P<0.01); (4) under the same conditions of pulse duration, voltage, plasmid concentration, and cell density, the transfection rates were 12.520% and 4.634% at temperature of 25℃and 4℃, respectively. The difference between the higher transfection rate at 25℃and that at 4℃was extremely significant (P<0.01).3. Cell toxicity sensitivity experiment was carried out to determine the tolerance of EPGCs to G418, and the optimum concentration of G418 by fast selection. The EPGCs could survive for 15d,14d,10d,8d,5d and 3d at G418 concentration of 100μg/mL,200μg/mL,300μg/mL,400μg/mL,500μg/mL and 600μg/mL, respectively. The results of fast selection for EPGCs after transfection for 48h in the concentration of 400μg/mL G418 medium indicated that: positive EPGCs clones did not appear until the sixth day, and all of the negative cells died until the eighth day. 4. EPGCs were transfected through electroporation with EGFP as report gene . The 112 chicken blastoderms were all dead at the fifteenth day, which injected with EPGCs after selection for 8 days. The whole blastoderms of 1-7 days after hatch and the tissues of muscle, liver and heart of 8-15 days were detected by PCR, and the result were as follows: positive rate of whole blastoderms DNA were 52.63% and 31.58% at 1-3d and 4-7d, respectively; and they were 20.00%, 10.00%, and 5.56% at 8-11 days, 12-13 days, and 14-15 days, respectively. It could indicated that the positive rate reduced along with hatch days.5. The fluorescence expression extent in muscle, liver and heart was different according to freezing tissue slices detection. it was higher in liver and heart than that in muscle.The result showed that it was feasible to produce transgenic chickens through EPGCs.
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