Study On Genetic Modification Of Chicken PGCs Mediated By Transposons

Primordial germ cells(PGCs)is now the main host cell in the research of transgenic chicken.PGCs are precursor cells of sperm and occyte,originating from the epiblast blastoderm,migrating via germinal crescent,inside and outside circulation and finally reaching gonads,forming sperm and occyte.To investigate efficiency of transgenic chicken production mediated by transposon,the transposon vectors and transposase vector were injected into HH13-14 chicken embryos to transfect PGCs in vivo.The gene transfer efficiency of different transposons and specific gene regulators were compared by testing reporter gene GFP expression level.The following results were obtained.1.The transposon vector pT3-PST-CAG-GFP and transposase plasmid were mixed at the mass ratio of 2:1,and injected into HH10 embryonic body.By detecting EGFP expression level of ED 14 chicken embryos,gene transfer efficiency of three transposons,SB,PB and Tol2,were compared.Experiment is divided into five groups:PB,SB,Tol2 group,opening-window group and non-window group.The results showed that the difference of survival rate of each group is not significant(P>0.05);PB,SB,Tol2 three transposons have higher efficiency of genetically modified in chicken and they can be mediated EGFP expression in chicken embryo such as gonad,intestine,stomach,brain et al.Difference between 3 groups of transgenic efficiency were not significant(P>0.05);Gonadal efficiency of SB group is higher than that of PB group,but there is no significant difference(P>0.05).Positive gonad of Tol2 group were not got.2.PGCs specific-expression promotor(PSP),SV40 Enhancer,and SB100X coding frame were cloned using high fidelity PCR method.Sequences were identified after DNA sequencing as 100%homology with original sequence.Using recombinant DNA methods,we successfully constructed PGCs specifically expressed vectors of pSV40En-PSP-GFP and pS V40En-PSP-SB 100X.In order to optimize the gene embedding medium,pSV40En-PSP-GFP with Lip2000CD and cationic polymer PEI were injected into HH13-14 chicken embryo blood vessels.Results illustrate that the difference of efficiency of genetically modified between Lip2000CD and PEI group was not significant(P>0.05),but PEI group of gonadal positive rate(6.67%)is slightly higher than Lip2000CD group(2.47%).In order to detect PSP expression features in PGCs specifically expressed,pT3-PST-CAG-GFP and pSV40En-PSP-SB100X or pCMV-SB100X were mixed at mass ratio of 2:1.The mixture was injected into HH13-14blood vessels of chicken embryo.Results showed that gonadal positive rate of PSP group(11.03%)is higher than CMV promoter group(1.47%).The PSP drive EGFP expressed specifically in the gonads,and expression strength of PSP group is stronger than CMV group.3.Nanos 3 'UTR and EGFP coding frame were cloned by high-fidelity PCR method.Sequences were identified after DNA sequencing as 100%homology with original sequence.Using recombinant DNA methods,pTNT-GFP-Nanos and pTNT-SB100X-Nanos were successfully constructed to transcript mRNA in vitro.In order to detect Nanos 3 'UTR biological characteristics,pT3-PST-CAG-GFP DNA and SB100X-Nanos mRNA or SB100X mRNA were co-injected to HH13-14 chicken embryo blood vessels.Preliminary results showed that EGFP were expressed in the gonad,live,heart and spleen of ED14 chicken embryo.This suggested that Nanos 3 'UTR guidance characteristics of the foreign gene enrichment in germ cells was not significant.The experiments conducted in this report showed that the transposon vectors containing PGCs specific elements pSV40En-PSP-SB100X/pTNT-SB100X-Nanos were successfully constructed.Through injecting to blood vessles,the transposon vector with PSP element was expressed specifically in PGCs,improved efficiency of PGCs genetically modified.This research provide a foundation for the development of transgenic chicken bioreactor.