RESUMEN
Expression of the bacterial beta-galactosidase reporter gene (lacZ) in the vector used for the Knockout Mouse Project (KOMP) is driven by the endogenous promoter of the target gene. In tissues from KOMP mice, histochemical staining for LacZ enzyme activity can be used to determine gene expression patterns. With this technique, we have produced a comprehensive resource of gene expression using both whole mount (WM) and frozen section (FS) LacZ staining in 313 unique KOMP mutant mouse lines. Of these, â¼ 80% of mutants showed specific staining in one or more tissues, while â¼ 20% showed no specific staining, â¼ 13% had staining in only one tissue, and â¼ 25% had staining in >6 tissues. The highest frequency of specific staining occurred in the brain (â¼ 50%), male gonads (42%), and kidney (39%). The WM method was useful for rapidly identifying whole organ and some substructure staining, while the FS method often revealed substructure and cellular staining specificity. Both staining methods had >90% repeatability in biological replicates. Nonspecific LacZ staining occurs in some tissues due to the presence of bacteria or endogenous enzyme activity. However, this can be effectively distinguished from reporter gene activity by the combination of the WM and FS methods. After careful annotation, LacZ staining patterns in a high percentage of mutants revealed a unique structure-function not previously reported for many of these genes. The validation of methods for LacZ staining, annotation, and expression analysis reported here provides unique insights into the function of genes for which little is currently known.
Asunto(s)
Regulación de la Expresión Génica/genética , Genes Reporteros/genética , Operón Lac/genética , Regiones Promotoras Genéticas/genética , Animales , Atlas como Asunto , Femenino , Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Coloración y Etiquetado , Relación Estructura-ActividadRESUMEN
Apolipoprotein E (Apoe)-deficient knockout mice were used to test the hypothesis that mutant mice preserved as evaporatively dried (ED) spermatozoa, stored at -80â°C for 6 months, and then recovered by ICSI will exhibit the same phenotype as before preservation. The birth rate of mice recovered by ICSI of evaporatively dried spermatozoa was lower than that of fresh spermatozoa (17.5 vs 38.0%). Progeny of mice preserved using evaporatively dried spermatozoa were reproductively sound. From these, the second generation of mice produced by natural mating showed lesions typical of APOE deficiency, including severe hypercholesterolemia, hypertriglyceridemia, markedly increased plasma low-density lipoprotein level, and extensive and severe atherosclerotic lesions in the aorta. We conclude that the expected phenotype caused by an induced genetic mutation can be faithfully recapitulated and sustained in subsequent generations of mice preserved and stored as ED spermatozoa and recovered using ICSI. Because it is simpler, faster, and cheaper than conventional (cryopreservation) and nonconventional (freeze-drying) preservation procedures, evaporative drying is a viable, cost-effective, and efficient method for preserving and storing valuable mutant mouse strains.