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Genetic manipulation of mouse embryonic stem cells by mutant λ integrase

✍ Scribed by Nicole Christ; Peter Dröge

Book ID
102843513
Publisher
John Wiley and Sons
Year
2002
Tongue
English
Weight
769 KB
Volume
32
Category
Article
ISSN
1526-954X

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✦ Synopsis

Abstract

Summary: Mutant λ integrases catalyze site‐specific recombination reactions inside mammalian cells. Here we demonstrate that the integrase system can be used to eliminate resistance marker genes from the genome of mouse embryonic stem cells. So‐called integrative and excisive recombination pathways led to the precise deletion of the neomycin gene, which was inserted together with a flanking pair of directly repeated recombination sites into the ROSA26 locus by standard targeting techniques. The excision of the resistance gene led to the expression of enhanced green fluorescence protein, which served as a means to sort out cells that had undergone site‐specific recombination. Southern analysis and DNA sequencing confirmed that strand exchange reactions had occurred in the genome as expected. Hence, the integrase system may be used in conjunction with other site‐specific recombinases as a tool in genome manipulation protocols. genesis 32:203–208, 2002. © 2002 Wiley‐Liss, Inc.

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