A new mouse mutant for the LDL receptor identified using ENU mutagenesis

Abstract

In an effort to discover new mouse models of cardiovascular disease using N-ethyl-N-nitrosourea (ENU) mutagenesis followed by high-throughput phenotyping, we have identified a new mouse mutation, C699Y, in the LDL receptor (Ldlr), named wicked high cholesterol (WHC). When WHC was compared with the widely used Ldlr knockout (KO) mouse, notable phenotypic differences between strains were observed, such as accelerated atherosclerotic lesion formation and reduced hepatosteatosis in the ENU mutant after a short exposure to an atherogenic diet. This lossof-function mouse model carries a single base mutation in the Ldlr gene on an otherwise pure C57BL/6J (B6) genetic background, making it a useful new tool for understanding the pathophysiology of atherosclerosis and for evaluating additional genetic modifiers regulating hyperlipidemia and atherogenesis. Further investigation of genomic differences between the ENU mutant and KO strains may reveal previously unappreciated sequence functionality. The incidence and progression of atherosclerosis remains a challenge for public health intervention and medical research strategies. Mouse models have been of great utility in meeting this challenge and have enabled systematic evaluation of genetic and environmental influences on the pathogenesis of this disease. Lipoproteins and their receptors play crucial roles in cholesterol homeostasis and, when functionally impaired, can accelerate atherogenesis. Mice with targeted mutations resulting in loss of function and those engineered for overexpression of apolipoproteins, their receptors, and key enzymes in lipid metabolism have been used extensively to investigate the complex etiology of atherosclerosis and to develop effective approaches to treatment (1-6). In addition to genetically engineered mouse models, chemical mutagenesis using N-ethyl-N-nitrosourea (ENU), which causes primarily single-nucleotide mutations, has produced numerous new mouse models for studying human disease. Many of these mutants carry a novel functional mutation in a known gene (7-11). For known genes, having multiple models with a variety of unique mutations allows a survey of phenotypic differences and functional annotation of genes and their products. Causative mutations in numerous ENU mutants identified from large-scale mutagenesis programs worldwide, collectively representing a broad spectrum of disease-related phenotypes, have yet to be identified, and still hold promise of revealing novel genes.Here we describe the identification and initial characterization of a new mouse model of high cholesterol and atherosclerosis generated using ENU mutagenesis and high-throughput phenotyping. Candidate gene sequencing identified a point mutation in the LDL receptor (Ldlr) gene. Comparison of this mutant with the well-characterized Ldlr knockout (KO) mouse model reveals certain phenotypic differences and supports it as a new tool for the study of familial hypercholesterolemia (FH) and atherosclerosis. METHODS Mice and husbandryNonmutagenized C57BL/6J (B...

Overexpression of tissue-nonspecific alkaline phosphatase (TNAP) in endothelial cells accelerates coronary artery disease in a mouse model of familial hypercholesterolemia

“…Mice harboring a point mutation in the ldlr gene resulting in C699Y amino acid substitution (C57BL/6J- Ldlr Hlb301 /J; WHC for “wicked high cholesterol” [22]) were obtained from The Jackson Laboratory (Bar Harbor, ME, USA; stock 5061). WHC were characterized in our laboratory in comparison with their parental strain C57BL/6J (Data Supplement).…”

“…Therefore, these mice were genotyped by BglI restriction analysis of exon 14 PCR products as described [22]. Hprt ALPL were genotyped as previously described [10].…”

Hypoxia-Inducible Factor Prolyl 4-Hydroxylase-2 Inhibition Protects Against Development of Atherosclerosis

“…The Ldlr Hlb301 mice were generated with chemical ethylnitrosourea mutagenesis, and their genome has not been fully characterized for potential additional mutations. 18 These Ldlr than that of the Ldlr Hlb301 mice ( Figure 5G), and the area of the necrotic core was likewise reduced by >60% ( Figure 5H), whereas no difference was seen in the amount of plaque collagen ( Figure IIE in the online-only Data Supplement). The number of WAT macrophage aggregates correlated positively with plaque sizes in the whole aorta and at the aortic origin, suggesting that also in this model reduced WAT inflammation associated with atherosclerosis protection ( Figure 5I).…”

“…n=7 per group. 18 and obtained live double gene-modified pups. The Ldlr Hlb301 mice were generated with chemical ethylnitrosourea mutagenesis, and their genome has not been fully characterized for potential additional mutations.…”

Approach to assessing determinants of glucose homeostasis in the conscious mouse

“…ENU-induced mutagenesis is a strong method in that it can generate a wide array of subtle phenotypes and the resulting lines may more closely recapitulate the human phenotype compared to engineered knock-out models. Diet challenges are now being employed in this model to identify genetic and environmental interactions in the pathogenesis of metabolic disease (Lee et al 2012; Svenson et al 2008). …”