Unit

UNIT 23.5 Production of a Heterozygous Mutant Cell Line by Homologous Recombination (Single Knockout)

  1. Richard Mortensen

Published Online: 1 MAY 2001

DOI: 10.1002/0471142727.mb2305s52

Current Protocols in Molecular Biology

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.

Author Information

  1. University of Michigan Medical School, Ann Arbor, Michigan

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: OCT 2000

This is not the most recent version of the article. View current version (1 APR 2008)

Abstract

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.