A lacZ reporter gene expression atlas for 313 adult KOMP mutant mouse lines

Abstract

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, ∼80% of mutants showed specific staining in one or more tissues, while ∼20% showed no specific staining, ∼13% had staining in only one tissue, and ∼25% had staining in >6 tissues. The highest frequency of specific staining occurred in the brain (∼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.[Supplemental material is available for this article.]

X-ray 3D imaging of gene expression in whole-mount murine brain by microCT, implication for functional analysis of tRNA endonuclease 54 gene mutated in pontocerebellar hypoplasia

“…Overall, a good agreement between regions with elevated densities detected by microCT and the X-gal/FeCN chromogenic precipitates, product of lacZ reaction, was observed when the forebrain 2D sections were analysed ( Figure 2B,C). Importantly, histological analysis of control samples did not reveal any chromogenic product of lacZ activity in the brains of the wild type littermates (data not shown) and this result is in agreement with previously published data (42).…”

“…BioRxiv galactopyranoside) as a substrate for enzymatic detection of -galactosidase expression in combination with potassium ferri-and ferro-cyanide (X-gal/FeCN protocol) as suggested by IMPC consortium (www.kompphenotype.org) (42). In our experiments, the blue precipitate product of X-gal/FeCN staining is strong and visible in whole brain preparations from theTsen54-lacZ mice, but not in the wild type controls ( Figure 1B, C).…”

Lysine desuccinylase SIRT5 binds to cardiolipin and regulates the electron transport chain

“…Whole-mount staining was performed using the protocol of West et al (30). A liver lobe was freshly harvested, washed in PBS, fixed for 30 min, washed again, and then stained with X-Gal at 37°C for 4 h. The tissue was then viewed/photographed using a dissecting microscope (Leica).…”

A resource of targeted mutant mouse lines for 5,061 genes

“…Subsequently, large-scale mouse production and phenotyping programs deployed these unique resources, establishing the feasibility of genome-scale mouse production and phenotyping [7][8][9] . Building upon these successes, the International Mouse Phenotyping Consortium (IMPC) was established to coordinate a network of programs around the globe, assuring uniformity and reproducibility of these efforts, including standardization of phenotyping protocols and the use of a single inbred mouse strain background, C57BL/6N, with the ultimate goal of generating and phenotyping a single-gene knockout (KO) mouse line for every protein-coding gene in the genome.…”