Developing of inducible cell lines for study gene gain of function

Many biological problems are amenable to comparative gain of function experimental approaches. Conventional approach by introducing expression vector at random integration sites are confounded by expression variation. Besides in mouse embryonic stem cells, gene targeting by homologous recombination is not possible in most cell lines. We developed novel expression system based on conditional Tet-on regulation with selectable Cre/Lox cassette exchange in a single-copy integration vector. We demonstrated method for generation of cell lines with highly efficient inducible cassette exchange target loci. This system can be introduce in cell lines despite of their origin, differentiation stage or proliferation ability. Our system is ideal for comparative gain of function studies, such as comparing phenotypic effects of various genes, or evaluating mutants of a given gene. The conditional nature of the expression system facilitates work with genes with toxic phenotypes that would affect generation of stable cell lines. The most important is to generate clonal cell line in which there is single inducible locus, which is expressed robustly, it is not silenced, and it is not leaky. In this presentation we will show successful generation of various inducible cell lines, including human embryonic stem cell line, and we will discuss the possibility of using our system for modifying various cancer cell lines.