The Generation Of Transgenic Chickens Integrated With PiggyBac And PBase Used For Constructing Mutation Library

The piggyBac transposon comes from cabbage looper,which has many advantages,such as high efficiency of transposition,large capacity of foreign gene,no overproduction inhibition and "local hopping".It was now used as an insertional mutagen in several organisms,such as fungi,protozoa,plants and vertebrates.Gene trap is an advantageous technique in generating gene mutations massively,which is beneficial to identify the functions of large quantities of genes.When gene trap vector inserts into the genome,it would generate a fusion mRNA of the reporter gene and the endogenous gene.The transcription of the endogenous gene was inhibitied by the insertional mutation.Transposon could induce gene trap vectors to integrate into the genome,which is available to generate insertional mutation among the whole genome.It has been widely used in transgene research among fruit flies,zebrafish and mice.This technique could be uesd for generating mutant and discovering new functional genes.Chicken is bred all over the world and plays an important role in the human life and scientific research.It has a special development mode.The chick embryo in a new laid egg has already developed in vivo,which contains about 60000 cells.It is difficult to produce transgenic chickens with the conventional methods used in mammal.In this study,we injected the piggyBac transport systems and the lentiviral vector expressing transpose separately into the subgerminal cavity,in order to produce the transgenic chickens integrated with piggyBac and PBase.We co-transfected the donor vector?pPB-IRES-GFPneo or pZGs?with a help vector?pCAG₄BP?into sub-germinal cavity of newly laid eggs to produce chimeric chickens.The PCR analysis of the heart,liver,and gonad of the offspring showed that piggyBac had inserted into the genome,and the chimerism of different tissues was different.We selected five cocks with gonadal mosaicism to generate G1 chickens.By the means of PCR,we obtained eight heterozygous individuals from 435 offspring,and the positive rate was about 1.8%.Southern Blot analysis showed the transgene was single copy.Genome walking result of the Gl generation A13 demonstrated that the transposon inserted into intergenic region.The reporter gene could not express for the vector did not trap the endogenous genes.On the other hand,we produced transposase chimeric chickens by lentivirus method.We injected 185 fertilized eggs,and obtained 55 chicks.The average hatching rate was 29.7%.The detection of the whole embryos and the tissues from the heart,liver,and gonad showed that the chimerism was different in offspring.We got one rooster with gonadal mosaicism.After passaging,we have obtained two heterozygous individuals with single copy by the detection of PCR and Southern Blot.The test of genome walking showed that the insertional sites were in the introns of different genes.In our research,we have successfully generated the two transgenic chickens integrated with piggyBac and PBase,which could be used for further study.When these two kinds of chickens get sexual maturity,researchers could artificially cross them and screen the progenies containing new insertional sites,in order to make a mutation library in chickens.This will provide theoretical guidance and study objects for finding new functional genes.