Knockout Mice as an Experimental Model to Study the Biology of Inositol Pyrophosphates.

Knockout mice have served as excellent model systems to investigate the functions of specific mammalian proteins at the organismal level. Several studies on tissue-specific or whole-body depletion of individual IP6 kinase (IP6K) paralogs have shed light on myriad roles for their product, the inositol pyrophosphate 5-InsP7, in different physiological processes and pathological states. The loss of Ip6k1, Ip6k2, or Ip6k3 leads predominantly to nonoverlapping phenotypes in mice, reflecting their differential tissue, cell type, and subcellular distribution. Using the example of Ip6k1 knockout mice, and the stark phenotype of male infertility resulting from the depletion of condensed spermatids in the testes of these mice, this chapter provides detailed methods for the use of knockout mice as a model to study the functions of IP6Ks. We begin with protocols for the maintenance and breeding of the mouse colony, with instructions to genotype offspring from the mating of heterozygous mice carrying one Ip6k1 knockout allele. We then provide methods for the histopathological comparison of tissues in wild-type versus knockout mice by hematoxylin and eosin staining, and the detection of IP6K1 expression in specific tissues and subcellular compartments by western blotting and immunofluorescence.