Faculty Collaboration Database

PCR-based gene targeting of the inducible nitric oxide synthase (NOS2) locus in murine ES cells, a new and more cost-effective approach. Transgenic Res 1996 Nov;5(6):413-20

Date

11/01/1996

Pubmed ID

8840524

DOI

10.1007/BF01980206

Scopus ID

2-s2.0-0030298386 (requires institutional sign-in at Scopus site)   6 Citations

Abstract

Gene targeting by double homologous recombination in murine embryonic stem (ES) cells is a powerful tool used to study the cellular consequences of specific genetic mutations. A typical targeting construct consists of a neomycin phosphotransferase (neo) gene flanked by genomic DNA fragments that are homologous to sequences in the target chromosomal locus. Homologous DNA fragments are typically cloned from a murine genomic DNA library. Here we describe an alternative approach whereby the inducible nitric oxide synthase (NOS2) gene locus is partially mapped and homologous DNA sequences obtained using a long-range PCR method. A 7 kb NOS2 amplicon is used to construct a targeting vector where the neo gene is flanked by PCR-derived homologous DNA sequences. The vector also includes a thymidine kinase (tk) negative-selectable marker gene. Following transfection into ES cells, the PCR-based targeting vector undergoes efficient homologous recombination into the NOS2 locus. Thus, PCR-based gene targeting can be a valuable alternative to the conventional cloning approach. It expedites the acquisition of homologous genomic DNA sequences and simplifies the construction of targeting plasmids by making use of defined cloning sites. This approach should result in substantial time and cost savings for appropriate homologous recombination projects.

Author List

Randolph DA, Verbsky JW, Yang L, Fang Y, Hakem R, Fields LE

Author

James Verbsky MD, PhD Professor in the Pediatrics department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Chromosome Mapping
Chromosomes
Embryo, Mammalian
Genetic Vectors
Mice
Mutagenesis, Site-Directed
Nitric Oxide Synthase
Polymerase Chain Reaction
Recombination, Genetic
Stem Cells
Transfection