Method for integrating genes at specific sites in mammalian cells via homologous recombination and vectors for accomplishing the same

摘要

Provided is a method for inserting DNA at a target site in the genome of a mammalian cell by: 1 transfecting or transforming a mammalian cell with a marker plasmid containing the following sequences: 1.1 a region of DNA that is heterologous to the mammalian cell genome which when integrated in the mammalian cell genome provides a unique site for homologous recombination, 1.2 a DNA fragment encoding a first inactive portion of a first selectable marker protein, and 1.3 at least one other selectable marker DNA that provides for selection of mammalian cells which have been successfully integrated with the marker plasmid; 2 selecting a cell which contains the marker plasmid integrated in its genome by screening for expression of the at least one other selectable marker DNA of step 1.3; 3 transfecting or transforming the selected cell with a target plasmid which contains the following sequences: 3.1 a region of DNA that is identical or is sufficiently homologous to the unique region in the marker plasmid such that this region of DNA can recombine with the DNA via homologous recombination, 3.2 a DNA fragment encoding a second inactive portion of the first selectable marker contained in the marker plasmid, wherein the active selectable marker protein encoded by the DNA is only produced if the fragment is expressed in association with the fragment of the selectable marker DNA contained in the marker plasmid; and 4 selecting cells which contain the target plasmid integrated at the target site by screening for the expression of the first selectable marker protein.