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Introduction:Marker-free transgenic technology is highly desired to create genetically modified pigs,in term of food safetyand consumer ethics.AΦC31 integrase has been used to introduce site-specific transgene integration into pseudo attPsites of animal genomes.It represents a powerful tool to produce transgenic animals.Here we attempted to examineits efficacy to mediate transgene integration into pig genome.We also wished to establish a marker-free transgenictechnology in large farm animal.Materials and methods:Donor and acceptor pigs were Hubei White Pig raised in the farm of Institute of Animal Science and VeterinaryMedicine,Hubei Academy of Agricultural Science.PBCPB+and PCMV-1NT were gifts of Prof.Calos(StanfordUniversity).PEGFP-N1-attB reporter plasmid was constructed in our group.ΦC31 mRNA were purchased fromStanfordbio Inc.(Jiangsu).Results and discussion:ΦC31 mRNA and attB-containing pEGFP-N1 plasmid(PEGFP-N1-attB)were microinjected into 120 pig 1-cellzygotes and transferred into 5 surrogate pigs.32 piglets were live born from 4 acceptor pigs.PCR analysis showed that 7 of them were attB and EGFP positive individuals.The transgenic positiverate is 7/32=22%;gene transfer efficiency is 7/120=5.83%.In comparison to naked DNA pronuclearmicroinjection,this efficiency is significantly high.One hot spot pseudo attP site (pig pseudo attP-1,ppp-1)was identified from2 piglets by TAIL-PCR.PPP-1 is located in an intergenic region of pig chromosome 1,implyingthat transgene integration into this site does not destroy any endogenous genes.Up to now no abnormal phenotypeswere observed in term of birth weight,animal behaviors.This also demonstrated that PPP-1 appears to be safeharbor for transgene integration.