Patent ID: 7473557
Filing Date: 2009-01-06
Classification: A01K,C12N

Abstract:
1. A method of targeting a selection cassette into a transcriptionally active gene in a mouse embryonic stem (ES) cell, comprising: (a) constructing a DNA targeting vector consisting of, in order, (1) a 5′ homology arm, (2) a promoterless selection cassette comprising, in order, a splice acceptor sequence, a selectable marker gene and a first polyadenylation signal sequence, wherein the selectable marker gene and first polyadenylation signal sequence are flanked on each side by a site-specific recombinase recognition sequence, (3) a gene of interest and a second polyadenylation signal sequence, and (4) a 3′ homology arm, wherein the 5′ and 3′ homology arms each display homology to a sequence of the transcriptionally active gene, and wherein upon homologous recombination the promoterless selectable marker gene is operably linked to the promoter of the transcriptionally active gene, and the gene of interest is operably linked to the promoter of the transcriptionally active gene following recombinase-mediated removal of the selectable marker; (b) introducing the DNA targeting vector of (a) into the ES cell; (c) selecting the ES cell of (b) for expression of the selectable marker gene, and (d) screening the selected ES cells of (c) to identify those cells in which the selection cassette has integrated into the transcriptionally active gene, wherein targeting frequency is 100%.