RESUMO
Nicotinamide nucleotide transhydrogenase (NNT) is an inner mitochondrial membrane transmembrane protein involved in regenerating NADPH, coupled with proton translocation across the inner membrane. We have shown that a defect in Nnt function in the mouse, and specifically within the beta-cell, leads to a reduction in insulin secretion. This chapter describes methods for examining Nnt function in the mouse. This includes generating in vivo models with point mutations and expression of Nnt by transgenesis, and making in vitro models, by silencing of gene expression. In addition, techniques are described to measure insulin secretion, calcium and hydrogen peroxide concentrations, membrane potential, and NNT activity. These approaches and techniques can also be applied to other genes of interest.
Assuntos
Insulina/metabolismo , Mitocôndrias/enzimologia , NADP Trans-Hidrogenases/genética , NADP Trans-Hidrogenases/metabolismo , Animais , Cálcio/análise , Linhagem Celular , Inativação Gênica , Peróxido de Hidrogênio/análise , Secreção de Insulina , Células Secretoras de Insulina/enzimologia , Células Secretoras de Insulina/metabolismo , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Mitocôndrias/genética , Mutação PuntualRESUMO
N-ethyl-N-nitrosourea (ENU) introduces mutations throughout the mouse genome at relatively high efficiency. Successful high-throughput phenotype screens have been reported and alternative screens using sequence-based approaches have been proposed. For the purpose of generating an allelic series in selected genes by a sequence-based approach, we have constructed an archive of over 4000 DNA samples from individual F1 ENU-mutagenized mice paralleled by frozen sperm samples. Together with our previously reported archive, the total size now exceeds 6000 individuals. A gene-based screen of 27.4 Mbp of DNA, carried out using denaturing high-performance liquid chromatography (DHPLC), found a mutation rate of 1 in 1.01 Mbp of which 1 in 1.82 Mbp were potentially functional. Screening of whole or selected regions of genes on subsets of the archive has allowed us to identify 15 new alleles from 9 genes out of 15 tested. This is a powerful adjunct to conventional mutagenesis strategies and has the advantage of generating a variety of alleles with potentially different phenotypic outcomes that facilitate the investigation of gene function. It is now available to academic collaborators as a community resource.