Generation of PDE4 knockout mice by gene targeting

Methods Mol Biol. 2005;307:191-210. doi: 10.1385/1-59259-839-0:191.

Abstract

The development of gene-targeting techniques has ushered in a new era in mouse genetics. Two discoveries have been instrumental: the finding that an exogenous DNA introduced in mammalian cells can recombine with homologous chromosomal sequences, a process known as gene targeting, and the revelation that cultured embryonic stem (ES) cells when injected into early stage mouse embryos can contribute to produce germ-line chimeras. On the basis of these seminal findings, gene targeting by homologous recombination in mouse ES cells in vitro has been established as a powerful means of altering specific loci in the mouse genome. As a result, gene function can be studied in vivo. By applying this technology, targeted disruption of PDE4 alleles is created in cultured ES cells and, subsequently, the mutant ES cells are injected into blastocysts and returned to pseudopregnant foster mothers to produce germ-line chimeric pups. In this chapter, we describe the basic protocols used to generate the PDE4 knockout mice.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3',5'-Cyclic-AMP Phosphodiesterases / genetics*
  • 3',5'-Cyclic-AMP Phosphodiesterases / metabolism
  • Animals
  • Cyclic Nucleotide Phosphodiesterases, Type 4
  • Embryo, Mammalian / cytology
  • Embryo, Mammalian / enzymology*
  • Gene Targeting* / methods
  • Mice
  • Mice, Knockout*
  • Recombination, Genetic*
  • Stem Cells / cytology
  • Stem Cells / enzymology*

Substances

  • 3',5'-Cyclic-AMP Phosphodiesterases
  • Cyclic Nucleotide Phosphodiesterases, Type 4