B6.Cg-Apoe^{tm3(APOE*4)Mae} Ins2^Akita Ldlr^tm1(LDLR)Mae/J

Stock number: 012628
Research areas: Cardiovascular Research, Cell Biology Research, Diabetes and Obesity Research, Endocrine Deficiency Research, Metabolism Research, Neurobiology Research, Research Tools

Description

In "E4hAkita" mice, the mouse apolipoprotein E is replaced with a human apoE4 isoform and the mouse low density lipoprotein receptor is replaced with a transcript-stabilized human LDLR that expresses at three times that of wildtype endogenous levels. Also present is the Ins2^Akita allele which causes insulin dependent diabetes. E4hAkita mice are diabetic with atherosclerosis and may be useful in applications related to the study of lipoprotein metabolism-related genes in diabetes, atherosclerosis, dyslipidemia, and vascular complications.

Detailed description

Apoe⁴ Ldlr^h Ins2^C96Y mice harbor two humanized knock-in mutations, the human APOE*4 replacement allele (Apoe^{tm3(APOE*4)Mae}) and the human LDLR replacement allele (Ldlr^tm1(LDLR)Mae), along with the dominant type 1 diabetes mutation Akita (Ins2^Akita). Unless noted otherwise, the phenotypes below are for mice maintained on regular chow (~5% fat; <0.1% cholesterol).

The human APOE*4 replacement allele (Apoe⁴ or E4) expresses the human apoE4 isoform in place of the endogenous apoE protein. Under direction of the endogenous mouse apoE promoter/regulatory regions, human apoE4 expression is observed at physiological levels in the same temporal and spatial pattern observed in humans. Mice homozygous for human APOE*4 (Apoe^4/4) are viable and fertile with normal plasma lipid and lipoprotein profiles. Apoe^4/4 show mild retinal changes with aging. When maintained on high fat/cholesterol diet mirroring that consumed by humans, Apoe^4/4 exhibit increased susceptibility to atherosclerosis, impaired glucose tolerance, fat overload, amyloid beta immunoreactivity, degenerative changes in the retina and other metabolic defects.

The human LDLR replacement allele (Ldlr^h) has the promoter/regulatory regions and exon 1 (encoding only the signal peptide) from endogenous mouse Ldlr directing expression of a human LDLR sequence modified to have increased mRNA transcript stability; this results in expression of hLDLR protein in place of endogenous mouse Ldlr protein, with three-fold greater levels. Both homozygous human LDLR (Ldlr^h/h) mice and heterozygous human LDLR (Ldlr^h/+) mice are viable and fertile with a ~40% reduction in steady state plasma cholesterol. The reduction is primarily in the HDL cholesterol fraction, resulting in elevated non-HDL/HDL cholesterol ratio. The HDL cholesterol of Ldlr^h/+ mice is <50 mg/dl, and is close to that normally seen in humans. Of note, because of the equivalency in protein expression and reduced HDL between heterozygotes and homozygotes, the Ldlr^h/+ genotype has been more extensively characterized.

The dominant type 1 diabetes mutation Akita (Ins2^C96Y) has a C96Y amino acid substitution in the insulin II gene; this results in improper folding of pro-insulin in the endoplasmic reticulum and causes eventual pancreatic islet beta-cell death. As a consequence, heterozygous (Ins2^C96Y/+) males develop insulin dependent diabetes around one month of age, including hyperglycemia, hypoinsulinemia, polydipsia and polyuria, without obesity or insulitis. Heterozygous males are more severely affected than females.

Stock No. 012307: Mice homozygous for human APOE*4 and heterozygous for human LDLR (Apoe^4/4 Ldlr^h/+) are called E4h mice. E4h mice are viable and fertile with low plasma cholesterol (normal non-HDL cholesterol) presumably due to increased Ldlr metabolism in the liver. E4h mice are highly sensitive to diet-induced dyslipidemia. Specifically, when maintained on high fat/cholesterol diet mirroring that consumed by humans, E4h animals develop hypercholesterolemia and atherosclerosis. The donating investigator reports mice homozygous for Apoe⁴ and homozygous for Ldlr^h are viable and fertile with no breeding problems.

Stock No. 012628: Mice homozygous for human APOE*4, heterozygous for human LDLR and heterozygous for Akita (Apoe^4/4 Ldlr^h/+ Ins2^C96Y/+) are called E4hAkita mice. E4hAkita mice are viable and fertile. The phenotype of E4hAkita males is described below. E4hAkita males develop spontaneous diabetes (by two months of age) and atherosclerosis. In addition, males are hyperphagic (but have reduced body weight) and hypoinsulinemic, with a modest but significant reduction of plasma triglycerides. Combining the Ldlr^h-induced elevation in non-HDL/HDL cholesterol ratio with the diabetes (Akita)-induced increase in small, cholesterol-enriched, apoB48-containing VLDL results in E4hAkita mice having a non-HDL/HDL cholesterol ratio (~3) that is increased ~6-fold compared to wildtype mice and very close to the human ratio (~3). E4hAkita mice exhibit distinct atherosclerotic foam cell lesions in aortic roots, despite having plasma total cholesterol within the normal physiological range (<120 mg/dl) observed for wildtype mice. Of note, no atherosclerosis is reported for apoE4 homozygotes alone (E4 mice), with hLDLR (E4h mice) or with diabetes (E4Akita mice).

Development

Apoe⁴ Ldlr^h Ins2^C96Y mice (or E4hAkita mice) harbor two humanized knock-in mutations, the human APOE*4 replacement allele (Apoe^{tm3(APOE*4)Mae}) and the human LDLR replacement allele (Ldlr^tm1(LDLR)Mae), along with the dominant type 1 diabetes mutation Akita (Ins2^Akita).

The human APOE*4 replacement allele (Apoe⁴, apoE4 or E4) targeting construct was designed by Dr. Nobuyo Maeda (University of North Carolina, Chapel Hill) to insert a DNA sequence from human apolipoprotein E (APOE) exons 2-4 (encoding the APOE*4 isoform) and a neomycin-resistant cassette, such that it replaced the equivalent portions of the mouse Apoe locus on the proximal end of chromosome 7. The construct was electroporated into BK4 embryonic stem (ES) cells (a subclone of the 129P2/OlaHsd-derived E14TG2a ES cell line). Correctly targeted ES cells were injected into recipient blastocysts, and chimeric animals were bred to C57BL/6 mice to establish the mutant colony. The Apoe⁴ colony was backcrossed at least six generations to C57BL/6 mice. The resulting B6.Apoe⁴ mice were then used as described below.

Ins2^C96Y (Akita or "maturity onset diabetes of the young 4") is a dominant spontaneous mutation with a G to A transition at nucleotide 1907 (encoding Cys to Tyr at amino acid 96) of the insulin II locus (Ins2) on the distal end of chromosome 7. Akita mice on a C57BL/6 background are available and described as Stock No. 003548. Dr. Maeda obtained B6.Akita mice and bred them as described below.

The human LDLR replacement allele (Ldlr^h or hLDLR) targeting construct was created by Dr. Nobuyo Maeda (University of North Carolina, Chapel Hill). First, a human low density lipoprotein receptor (LDLR) minigene was created to have a genomic sequence of human LDLR from exon 2 through part of exon 5, followed by a cDNA sequence of human LDLR from part of exon 5 through exon 18 that is modified to encode a more stable mRNA transcript (by deletion of two of the three 3' AU-rich elements in exon 18). The targeting vector, containing the human LDLR minigene followed by a human growth hormone polyA addition signal sequence and a neomycin-resistant cassette, was designed to replace ~8 kbp of sequence spanning intron 1 through most of exon 4 of the mouse Ldlr locus on chromosome 9. The construct was electroporated into 129S6/SvEvTac derived TC-1 ES cells. Correctly targeted ES cells were injected into recipient blastocysts, and chimeric animals were bred to C57BL/6J mice to establish the mutant colony. The Ldlr^h colony was backcrossed at least six generations to C57BL/6 mice. The resulting B6.Ldlr^h mice were then used as described below.

To generate the triple mutant line, Dr. Maeda first bred B6.Apoe⁴ mice with B6.Akita mice to identify a recombination event. These mutations are located ~122 Mbp apart on chromosome 7. The double mutant mice were sent to The Jackson Laboratory Repository in 2010. Upon arrival, they were bred with C57BL/6-congenic Apoe⁴ Ldlr^h animals (Stock No. 012307). The resulting triple mutant mice, Apoe⁴ Ldlr^h Ins2^C96Y mice are Stock No. 012628. Mice homozygous for human APOE*4, heterozygous for human LDLR and heterozygous for Akita (Apoe^4/4 Ldlr^h/+ Ins2^C96Y/+) are called E4hAkita mice.

Allele

Symbol: Ins2^Akita
Name: Akita
MGI accession ID: MGI:1857572
Generation method: Spontaneous
Marker symbol: Ins2
Marker name: insulin II
Chromosome: 7
Strain of origin: C57BL/6NSlc
Molecular note: In the mutant allele a transition from G-to-A at coding nucleotide 287 disrupts an Fnu4HI site in exon 3. This mutation changed the seventh amino acid in the A chain of mature insulin, Cys96 (TGC), to Tyr (TAC) (p.C96Y). The authors predict that the transition would disrupt a disulfide bond between the A and the B chains and would likely induce a major conformational change in insulin 2 molecules. RT-PCR studies suggest that both normal and mutant Ins2 alleles are transcribed similarly in pancreatic islets of heterozygous mice, although immunofluorescence and immunoblot analyses of heterozygous islets detected reduced levels of insulin and proinsulin.

Allele

Symbol: Ldlr^tm1(LDLR)Mae
Name: targeted mutation 1, Nobuyo Maeda
MGI accession ID: MGI:3691296
Generation method: Targeted
Attributes: Inserted expressed sequence; Humanized sequence
Marker symbol: Ldlr
Marker name: low density lipoprotein receptor
Chromosome: 9
Strain of origin: 129S6/SvEvTac
Expressed genes: LDLR,low density lipoprotein receptor,human
Site of expression: Plasma.
Molecular note: About 8 kb of sequence spanning 3' of intron 1 through exon 4 was replaced with the human LDLR minigene containing exons 2-18 of human sequence. The minigene was shortened by a deletion of two of the three 3' AU-rich elements in exon 18. The mature protein transcribed from the chimeric gene was entirely human protein, due to exon 1 encoding only the signal peptide.

Allele

Symbol: Apoe^{tm3(APOE*4)Mae}
Name: targeted mutation 3, Nobuyo Maeda
MGI accession ID: MGI:2158398
Generation method: Targeted
Attributes: Inserted expressed sequence; Humanized sequence
Marker symbol: Apoe
Marker name: apolipoprotein E
Chromosome: 7
Strain of origin: 129P2/OlaHsd
Expressed genes: APOE,apolipoprotein E,human
Site of expression: Blood plasma.
Molecular note: A DNA fragment containing exons 2-4 of the human APOE gene (the APOE4 isoform) replaced an equivalent portion of the mouse Apoe gene. Western blot analysis on plasma derived from homozygous mice demonstrated that the human protein was expressed from this allele and the endogenous mouse protein was not detectable.