Construction Of Transgenic Swine Of Specific Expressing Cre Recombinase Controlled By PIWIL1Promoter

Non-coding RNA mainly including siRNA (small interfering RNA)、miRNA(microRNA) and piRNA (PIWI-interacting RNA), piRNA is the hot researcharea which scientists continue pay attention to. piRNA was found ingerm cells ofanimals (Drosophila melanogaster, rats, mice and human), it is a kind of novel smallmolecules of endogenous non-coding RNA. piRNAs are found in clusters throughoutthe genome,but it is highly discontinuous, the length of a piRNA is, by definition,between26and32nucleotides, because of its specifical interaction with pIWIproteins, so piRNA is also known as PIWI-interacting RNA.piRNA gene clusters aremainly distributed in the region of transposons and repeat sequences， thechromosomal location of species is conservatism. piRNA mainly expressed in thegerm cell line represents a unique small RNA pathway, which enables the germ cellstransposon silencing. They may be involved in germline stem cell self-sustaining anddifferentiation fate decision, meiosis, sperm formation and other reproductive processthrough epigenetic and post-transcriptional regulation.They play an important role inMaintaining germline DNA integrity, inhibiting the transcription of the transposon,inhibiting translation, involving in heterochromatin formation, implementing ofepigenetic regulation and germ cells cytogenesis.The Argonaute (AGO) protein family is divided into two major subfamilies,Theyare the AGO subfamily similar to Arabidopsis thaliana Argonautel and PIWI proteinsubfamily similar to Drosophila PIWI. There are eight members in Human AGOprotein family by sequence comparing, they are4eIF2C/AGO subfamily members(EIF2C1/hAGO1, EIF2C2/hAGO2EIF2C3/hAGO3and EIF2C4/hAGO4), as well asthe four members of PIWI subfamily (PIWIL1/HIWI, PIWIL2/HILI, PIWIL3, andPIWIL4/HIWI2)[8]. The four members of eIF2C/AGO subfamily are expressed invarious tissues in the adult，including testis, heart, kidney, thymus.and four membersof the PIWI subfamily are expressed in the testis of spermatogonia or spermatocytes.AGO protein subfamily and PIWI protein subfamily can bind with non-coding small RNA, in transcription, and play a key role in regulating target genes andproteins in transcription or translation level [9-10]. The Argonaute protein family arethe key factors of the RNA silencing pathway [11], PIWI members are along with thepiRNA in the biogenesis and the exercise of the biological functions of the processand silence transcription of genes, including participation in the process ofmaintaining the germ system and stem cell function and regulation of translation andmRNA stability.Although there are extensive research for the piRNA and we have achievedinitial results, there are still many problems to make further exploring, such as piRNAand PIWI protein interaction mechanism and regulation. Some studies have reportedthat PIWI protein has a high expression in non-reproductive organs, suggesting thatpiRNA may also have an important role in the regulation in other tissues besides playan important role in the process of gamete development.In this experiment, the background is the above-mentioned researchresults.Amplify the promoter of pig PIWIL1gene by PCR. Replace the MHCpromoter of pig specific Cre recombinase expression plasmid (pET28a-MHC-Cre-BGHpo1yA-FRT2neor) with PIWIL1promoter and specific Crerecombinase plasmid(pPIWIL1-Cre) was abtained. Transfect the plasmid tomini-pig fetal fibroblast cells and screen for positive clones with drug and donuclear transfer. We generate transgenic pigs that specific express Crerecombinase successfully, lay a foundation for researching the function ofpiRNA, revealing the piRNA and PIWI protein interaction mechanism whichproviding a reliable animal platform.