Anindita A, Sen Jayraj, Bhandari Rashna
Laboratory of Cell Signalling, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India.
Graduate Studies, Manipal Academy of Higher Education, Manipal, India.
Methods Mol Biol. 2025;2972:249-266. doi: 10.1007/978-1-0716-4799-8_18.
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-InsP, 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.
基因敲除小鼠已成为在机体水平研究特定哺乳动物蛋白质功能的优秀模型系统。多项关于单个肌醇六磷酸激酶（IP6K）旁系同源物的组织特异性或全身敲除的研究，揭示了其产物肌醇焦磷酸5-InsP在不同生理过程和病理状态中的众多作用。Ip6k1、Ip6k2或Ip6k3的缺失在小鼠中主要导致不重叠的表型，反映了它们在组织、细胞类型和亚细胞分布上的差异。以Ip6k1基因敲除小鼠为例，以及这些小鼠睾丸中浓缩精子细胞缺失导致的明显雄性不育表型，本章提供了使用基因敲除小鼠作为模型研究IP6Ks功能的详细方法。我们首先介绍小鼠群体的饲养和繁殖方案，并指导对携带一个Ip6k1基因敲除等位基因的杂合小鼠交配后代进行基因分型。然后，我们提供通过苏木精和伊红染色对野生型与基因敲除小鼠组织进行组织病理学比较的方法，以及通过蛋白质免疫印迹和免疫荧光检测特定组织和亚细胞区室中IP6K1表达的方法。
