The Construction Of Rnai Technology In Chicken Embryo And Its' Preliminary Application On Function Study Of Dmrt1

This study will set up the RNA interference technology in chicken model,including shell windowing,transfecting chicken embryos with RNA-interference plasmid and detecting the interference effect,to found a platform for studying functions of sex-determine and sex-differentiation genes.The results were described as follows:1.We compared the survival rate of five-day chicken embryos of four teams:the common(team 1),windowing machinerily(team 2),Hydrochloric Acid Corrosion and remove the shell membrane(team 3),Hydrochloric Acid Corrosion and keep the shell membrane(team 4),the results were that the survival rate of team 2 is the lowest,only 14.29%;the survival rate of team 3 is the highest,about 77.27%:compared with the other three teams,the survival rate of five-day chicken embryos of team 2 is lowest,the difference is significant(|U|＞U_0.01);the difference between team 1 and team 3 is not significant,so did team 3 and team 4(|U|＜U_0.05);The results show that Hydrochloric Acid Corrosion method may reduce the effect of shell windowing to chicken embryos,and it's a safe,effective method for shell windowing.2.We compared three methods for transfecting the no-load RNA-interference plasmids into chicken embryos,by electroporation,liposome mediated and injecting directly.The results showed that the survival rate by electroporation is lower than the other two methods,the difference is significant(|U|＞U0.01);The difference between liposome mediated and injecting directly is not significant(|U|＜U0.05);The transfection efficiency of electroporation is higher than the other two methods(|U|＞U0.05),The difference between liposome mediated and injecting directly is not significant(|U|＜U0.05); And we found that the plasmid expressed in the heads of chicken embryos,also in the somites and the trails,when injecting the plasmids into the blood vessel of vitellicle,we conjectured that the plasmid may reach to other tissues by blood circulation:The plasmid only expressed in the area between the two electrodes,when transfecting by electroporation.Combining all of the matters,we transfected the embryos by electroporation,but the method of injecting plasmids into the blood vessel of vitellicle may be a good method for ulterior studies. 3.Detecting the expression of GFP at different times after tranfection.We detected the GFP in live organism imaging system when haven transfected for six to a hundred and twenty hours(interval six hours),and found that the GFP expressed twelve hours later after transfection,a hundred and twenty hours later,the GFP still expressed,and the plasmid expressed rate is the highest forty-eight hours later after transfection.4.We used DMRT1 gene to check effect of the technology that we set up.We firstly constructed an RNA-intefference plasmid aiming at DMRT1 gene,and transfected it into the three-day chicken embryos,using the windowing and transfection methods that we set up above;Collected live embryos with GFP at different time after transfection(five-day embryos,six-day embryos,seven-day embryos and eight-day embryos);selected three male and female embryos at every stage,so do the common team after sex identification. Extracted RNA of the embryos collected above,and transcript the RNA into cDNA, Expression of the DMRT1,AMH and CYP19A1 were studied by RT-PCR.Results showed that the expression level of DMRT1gene in male and female chicken embryos reduced significantly after transfection,compared with common teams,and in male embryos the expression level of AMH had a tendency of rising up in six-day embryos to eight-day embryos,so did the expression level of CYP19A1 in female embryos at different stage after transfection.We set up the technology of RNA interference in chicken embryo successfully,and it can be used in the studies of sex-determine and sex-related gene function.Meanwhile, the shell windowing method will provide meanwhile for the work of chicken embryos in vivo.