Construction Of A New Gene Targeting Vector PA2TR

In the method of gene targeting, exogenous gene fragment could be inserted in genome with homologous recombinase, which could change the gene locus exactly in the producing of transgenic animals and solve a series of problems with random insertion in the other methods. Construction of universal vector for gene targeting is very convenient for research in gene targeting. There are several kinds of vectors for gene targeting, but in those vectors there are small kinds of restriction enzymes sites, and the positive selective gene is still in the genome after targeting successfully, both of which restrict the uses of those vectors for gene targeting. In order to resolve those problems, in this research a new universal vector named pA2TR was made and its function was analyzed.1. Construction of universal vector for gene targeting and analysis of its functionPlasmid pGEM-3Z was used as the parent vetor ,inserting one neomycin gene (neo) for positive selection and two herpes simplex virus thymidine kinase gene tk1 and tk2 for negative selection in the multiple cloning site(MCS) of pGEM-3Z, inserting two locus of crossing-over(x) in P1(Loxp) in both side of neo ,to make a vector named pA2T. neo and tk1, neo and tk2 are separated by two different multiple cloning site(MCS) Neo,tk1and tk2 were translated separately. Transfection of pA2T into Goat fetus fibroblast cell with LIPOFECTAMINE 2000 conferred resistance to geneticin(G418) and resistance to ganciclovir(GAC) in the cell,These suggest the positive and negative selectable markers in the cell can express and thus the vector pA2T can be used as a universal gene targeting vector. Transformation of pA2T into BM25.8 which express Cre recombinase, conferred neo is deleted in the pA2T, This suggests the Loxp is active.2. Cloning gene of Cre recombinase and analysis of its expressionIn order to remove the positive selective gene after targeting successfully with Cre/LoxP system, the gene of Cre recombinase was cloned, vectors for prokaryotic and eukaryotic expression were made, and the activity of prokaryotic vector was tested.3. Cloning murine Oct-4 promoter and analysis of its functionMurine Oct-4 promoter was cloned and its activity in Goat fetus fibroblast cells was tested which suggested that the activity of Oct-4 promoter is silent in somatic cells.4. Construction of a new universal vector named pA2TR for gene targeting and analysis of its function The fragment of Oct-4 prmoter plus Cre recombinase was inserted in the vector pA2T, and a new vector named pA2TR was made. Analysis of its functions demonstrated that the vector could be used as a universal gene targeting vector.