Genetic knockouts and knockins in human somatic cells.
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.
Rago
Carlo
C
Sidney Kimmel Comprehensive Cancer Center, Cellular and Molecular Medicine Program, Johns Hopkins University School of Medicine, 1550 Orleans Street CRB2-453, Baltimore, Maryland 21231, USA.
Vogelstein
Bert
B
Bunz
Fred
F
D000483:Alleles; D018929:Cell Culture Techniques; D002460:Cell Line; D002478:Cells, Cultured; D000229:Dependovirus / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D011995:Recombination, Genetic