Genetic knockouts and knockins in human somatic cells

Research output: Contribution to journalArticle

Abstract

Gene targeting by homologous recombination with exogenous DNA constructs is the most powerful technique available for analysis of mammalian gene function. Over the past several years, the methods used to generate knockout and knockin mice have been modified for use in cultured human cells. The most significant innovation has been the adaptation of recombinant adeno-associated viruses (rAAVs) for such targeting. The stages of rAAV-mediated gene targeting include (i) the design and construction of a DNA targeting vector, (ii) the production of an infectious rAAV stock, (iii) the generation of cell clones that harbor rAAV transgenes, (iv) screening for homologous recombinants and (v) the iterative targeting of multiple alleles. The protocol described herein allows the generation of a cell line with a single altered allele in 3 months. A second allele of the same gene can be targeted in an additional 3 months.

Original languageEnglish (US)
Pages (from-to)2734-2746
Number of pages13
JournalNature protocols
Volume2
Issue number11
DOIs
StatePublished - Nov 15 2007

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ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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