UNIT 23.5 Production of a Heterozygous Mutant Cell Line by Homologous Recombination (Single Knockout)
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Molecular Biology
How to Cite
Mortensen, R. 2001. Production of a Heterozygous Mutant Cell Line by Homologous Recombination (Single Knockout). Current Protocols in Molecular Biology. 52:23.5:23.5.1–23.511.
- Published Online: 1 MAY 2001
- Published Print: OCT 2000
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Formerly unit 9.16, this unit takes a more appropriate place in Chapter 23, and has been updated and revised for this publication. Gene targeting by homologous recombination allows the introduction of specific mutations into any cloned gene. In this unit, the gene of interest is inactivated by interrupting its coding sequence with a positive selectable marker (e.g., neo). Expression of neo is obtained by including the phosphoglycerate kinase (PGK) promoter in the construct. To enrich for clones in which the target gene has undergone homologous recombination over those in which random integration of the construct has occurred, a negative selectable marker, herpes simplex virus thymidine kinase (HSV-TK), is included in the construct outside the region of homology to the target gene. Depending upon the target gene, it may be easier to assemble the construct by adding the neo and TK genes to the cloned target gene or by adding two fragments of the target gene to a plasmid containing the neo and TK genes.