Efficient site-directed dna integration into es and hematopoietic cells

Abstract

DNA mediated gene transfer to hematopoietic cells has been hindered by low gene transfer efficiency. In an attempt to achieve efficient, stable integration of potentially large DNA vectors at a site which confers expression in a defined and a predictable manner, we are exploiting a Cre/lox based recombination strategy. As an initial test in hematopoietic cells, murine BaF3 cells were used to introduce a hygromycin resistant gene flanked by 34 bp lox sites (floxed) using a retrovirus. Subsequently, co-electroporation of a floxed green flourescent protein (GFP) construct and the cre recombinase plasmid should mediate integration through the lox sites replacing hygromycin with GFP. Site directed, stable integration as confirmed by FACS and Southern blot analysis occurred at a high frequency ranging from 1–7% in 5 different BaF3 clones, or some 100 fold higher than the efficiency for a control GFP vector lacking lox sites. To assess if the expression characteristics of an initial integration site can be reutilized to express a transgene in a defined and predictable manner, floxed GFP expressing BaF3 clones were chosen that had clonally distinct levels of GFP expression that spanned over 300 fold. This differential expression was maintained following cre mediated replacement with a floxed cDNA human Thy-1 construct. Analogous findings were also obtained in the human K562 cell line. As an initial effort to extend this approach to primitive primary cells, we introduced lox sites into murine embryonic stem cells (ES) cells either by a retrovirus or by a plasmid. Subsequent cre mediated integration again occurred with high efficiency—up to 22% and clones retained in vitro differentiation capacity and germline competency. Site directed DNA integration should provide a powerful new approach for studying gene function and regulatory mechanisms in hematopoietic cells.