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KMID : 0578319940040010027
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Molecules and Cells
1994 Volume.4 No. 1 p.27 ~ p.32
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Gene Targeting in the Ke4 Locus of the Mouse in Embryonic Stem Cells
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Kang, Hae-Mook
Jacques, Benoit St./Schwartz, Faina/Shin, hee-Sup
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Abstract
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The mouse Ke4 gene was disrupted by targeting via homologous recombination in embryonic stem cells. Ke4 is a novel gene located in the H-2K region of the mouse and potentially encodes a transmembrane protein with histidine-rich charge clusters. To maximize the selection against the clones resulting from non-homologous recombination events, a targeting vector was devised that would allow the dominant selectable marker, the neomycin-resistance gene, to be conditionally expressed by in-frame fusion with the target gene product. Expression of the selectable marker from the vector can be accomplished in two ways: by homologous recombination into the target locus, that results in disruption of the target gene, or by random non-homologous integration near a promoter of another gene. The targeting vector was introduced into embryonic stem cells by electfoporation and G418-resistant clones were obtained. These clones were analyzed by Southern blots and polymerase chain reaction methods to examine whether they contain random or homologous integrations of the vector. Under ideal conditions, the targeting frequency on the Ke4 locus with this vector was as high as one in seven G418-resistant clones.
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