Establishment Of Oct4-eGFP Reporter System For Porcine Stem Cell Tracing

Pluripotent stem cells can both self-renew and differentiate into many other cell types, provide thebasis for developmental biology, cell therapy and gene modified research, domestic pig are ideal animalmodels for stem cell research. However, procine embryonic stem cells (ESc) have not yet established;there are still many problems in induced pluripotent stem cell (iPSc) research need to be solved.Establishing a reporter system useing cell pluripotency marker gene, can provide a valuable tool forthese studies. Oct4is an important cell pluripotency marker gene, which have been widely used foridentifying pluripotent cells in mice and other species, it can be used for establishing reporting systemfor stem cell tracing. Oct4-eGFP is an effective cells pluripotency visual reporter system，transgenicpigs could be of great value as a large animal model for studies of cell pluripotency, and could behelpful to establish procine ES cells and iPS cellsThis study successfully insert eGFP cDNA into endogenous Oct4gene at termination codon, theexpression of eGFP is under control of Oct4promoter, so expression of endogenous Oct4gene could bereflect by expression of eGFP. Oct4-eGFP transgenic cell lines of Wuzhishan miniature pig wereproduced via TALENs mediated homologous recombination. Oct4-eGFP cloned embryos wereproduced by Somatic cell nuclear transfer. Summary of the study is as follows:Study one: Customize TALENs and efficiency test. TALEN is a new tool for genomic editing,which can couse DNA double strand break, So as to induce gene mutation or improve the efficiency ofhomologous recombination. Ear fibroblast of Wuzhishan miniature pig was established, sequenceflanking termination codon of Oct4gene was acquired by sequencing,2pairs of TALENs targetingtermination codon of Oct4gene, which had subtle difference of DNA binding and cutting area, werecustom designed. Efficiency of this2pairs of TALENs were tested by sequencing of TALENs target site48h after transfection of TALENs vector and statistical targeted site sequence mutation rate.Theworking efficiency of the2pairs of TALENs were15%and13.2%.Study two: Construction of Donor DNA for gene knock-in.1.2kb sequence upstream of Oct4termination codon was amplified as left homologous arm,2A-eGFP gene was attched to lefthomologous arm by recombinant PCR;657bp sequence downstream of Oct4termination codon wasamplified as right homologous arm. The gene knock-in vector which based on homologousrecombination and positive-negative selection strategy was constructed. Neo resistant gene was betweenleft and right homologous arm as an positive screening markers, TK gene was in downstream of righthomologous arm as a negative screening markers. Two pairs of TALENs were respectively transfectwuzhishan miniature pig ear fibroblast with targeting vector. eGFP cDNA together with LoxP-flankedPGK-neo cassettes were insert into the Oct4gene of wuzhishan Miniature pig at the stop codon viaTALENs mediated homologous recombination.514drug-resistant cell clons were screened, and36correctly targeted clones were confirmed by PCR from them,the targeting efficiency was7%(36/514).Study three: removel of neo resistant gene and producting of transgenic cloned embryos. Correctly targeted clones were transient transfected with annular Cre recombinase vector plasmid to remove theneo resistant gene, PCR analys show that neo resistance gene were removed in some cells. Cells whichtransfected with Cre recombinase vector were used as nuclear donor for SCNT, without screening. PCRresults showed that neo resistance gene in some of the cloned embryos were removed.We observed theexpression of eGFP in cloned embryos at8cell stage and blastula stage, no obvious green fluorescencewere found in these cloned embryos, normal somatic cells cloned embryos were used as negativecontrol.The results show that TALENs mediated homologous recombination is suitable for large animalgenetic modification, This kind of method can greatly improve the efficiency of targeting, compareswith traditional homologous recombination. The positive and negative selection strategy based onhomologous recombination is an effective means for gene targeting. Construct gene targeting vectorwith Cre-LoxP system, selection markers can be deleted after positive cells were obtained.In conclusion, we successfully established technology platform for gene modification ofwuzhishan miniature pig via TALENs mediated homologous recombination, which provided a basis forsubsequent swine genetic modification research.