Construction of a new universal vector for insertional mutagenesis by homologous recombination

Abstract

We describe here the construction of a vector (pSSC-9) which can be used for the insertional mutagenesis of any gene for which genomic sequences have been cloned. This vector contains a neomycin-resistance-encoding gene (neoR) which is driven by a modified thymidine kinase (tk) promoter for positive selection. Flanking neoR are two tk genes driven by their own promoters for negative selection of nonhomologous insertions. The neoR and tk cassettes are separated by four unique cloning sites on the right-hand side of the neoR cassette and three unique sites on the left-hand side. The vector also includes two SfiI sites, one on each side of the tk cassettes, for the excision of the cloned genomic DNA fragments along with the selectable markers. Electroporation of pSSC-9 into mouse embryonic stem (ES) cells and cultured diploid mouse adrenal Y-1 cells conferred resistance to G418 and sensitivity to ganciclovir in both cell lines. These results illustrate the expression of the positive and negative selectable markers in two different cell lines and thus suggest that the vector could be used in ES cells, as well as in cultured somatic cells.