Production of Corticotropin‐releasing Hormone‐deficient Mice by Targeted Mutation in Embryonic Stem Cells

The generation of transgenic mice by introduction of intact or altered genes into the pronuclei of fertilized oocytes has proven a powerful tool in elucidating the consequences of ectopic or tissue‐appropriate overproduction of a given gene product; in defining DNA elements responsible for gene‐specific patterns of temporal, spatial, and regulated transcription; and in targeted oncogenesis for the development of cell culture lines for detailed in vitro study. This technology, however, has had limited success in addressing the role and essential function of specific peptides in the intact organism, except in the circumstance in which dominant negative alleles, capable of abrogating function of the normal allele, are available.1,2 With the establishment of embryonic stem (ES) cell culture systems amenable to in vitro manipulation for introduction of mutated genes, methods for selection of relatively rare homologous recombination events, and most importantly, the sustained capacity of these ES cells to contribute to the germline of chimeric mice, a method of addressing the consequences of loss of gene function has emerged.3,5 Targeted gene inactivation in ES cells with subsequent generation of deficient mice yields inheritable, complete loss of function at all stages of development and in all tissues. This is an advantage when assessing ontogenic importance, ability of interacting systems to compensate for loss of function, and the effects of chronic deficiency. These factors can also impair whole‐organism analysis of function, inasmuch as chronic deficiency may allow up‐regulation of compensatory systems, masking the in vivo function of the mutation, and global deficiency may make characterization at a specific site of synthesis difficult to determine. Most severely, loss of expression at all stages of development may lead to organism inviability.