RESUMEN
We describe here use of a cell-permeable Cre to efficiently convert the EUCOMM/KOMP-CSD tm1a allele to the tm1b form in preimplantation mouse embryos in a high-throughput manner, consistent with the requirements of the International Mouse Phenotyping Consortium-affiliated NIH KOMP2 project. This method results in rapid allele conversion and minimizes the use of experimental animals when compared to conventional Cre transgenic mouse breeding, resulting in a significant reduction in costs and time with increased welfare benefits.
Asunto(s)
Desarrollo Embrionario/genética , Integrasas/genética , Alelos , Animales , Cruzamiento , Embrión de Mamíferos , Células Madre Embrionarias , Ratones , Ratones TransgénicosRESUMEN
We report an albino C57BL/6N mouse strain carrying a spontaneous mutation in the tyrosinase gene (C57BL/6N-Tyr(cWTSI)). Deep whole genome sequencing of founder mice revealed very little divergence from C57BL/6NJ and C57BL/6N (Taconic). This coisogenic strain will be of great utility for the International Mouse Phenotyping Consortium (IMPC), which uses the EUCOMM/KOMP targeted C57BL/6N ES cell resource, and other investigators wishing to work on a defined C57BL/6N background.
Asunto(s)
Genoma , Ratones Endogámicos C57BL/genética , Monofenol Monooxigenasa/genética , Análisis de Secuencia de ADN , Albinismo/genética , Animales , Genómica , Genotipo , Ratones , Ratones Transgénicos , Monofenol Monooxigenasa/deficiencia , Monofenol Monooxigenasa/metabolismoRESUMEN
The Sanger Mouse Genetics Project generates knockout mice strains using the EUCOMM/KOMP-CSD embryonic stem (ES) cell collection and characterizes the consequences of the mutations using a high-throughput primary phenotyping screen. Upon achieving germline transmission, new strains are subject to a panel of quality control (QC) PCR- and qPCR-based assays to confirm the correct targeting, cassette structure, and the presence of the 3' LoxP site (required for the potential conditionality of the allele). We report that over 86 % of the 731 strains studied showed the correct targeting and cassette structure, of which 97 % retained the 3' LoxP site. We discuss the characteristics of the lines that failed QC and postulate that the majority of these may be due to mixed ES cell populations which were not detectable with the original screening techniques employed when creating the ES cell resource.
Asunto(s)
Células Madre Embrionarias/citología , Células Germinativas/citología , Ratones Mutantes/genética , Animales , Cruzamiento , Ratones , Control de CalidadRESUMEN
Heterozygous mutations in HNF1B in humans result in a multisystem disorder, including pancreatic hypoplasia and diabetes mellitus. Here we used a well-controlled human induced pluripotent stem cell pancreatic differentiation model to elucidate the molecular mechanisms underlying HNF1B-associated diabetes. Our results show that lack of HNF1B blocks specification of pancreatic fate from the foregut progenitor (FP) stage, but HNF1B haploinsufficiency allows differentiation of multipotent pancreatic progenitor cells (MPCs) and insulin-secreting ß-like cells. We show that HNF1B haploinsufficiency impairs cell proliferation in FPs and MPCs. This could be attributed to impaired induction of key pancreatic developmental genes, including SOX11, ROBO2, and additional TEAD1 target genes whose function is associated with MPC self-renewal. In this work we uncover an exhaustive list of potential HNF1B gene targets during human pancreas organogenesis whose downregulation might underlie HNF1B-associated diabetes onset in humans, thus providing an important resource to understand the pathogenesis of this disease.