Academic Senate - Meeting Minutes, 4/18/2017

<p>All values are presented with SD. Differences between <i>LDLR−/−</i> and the other two genotypes are significant where indicated, ANOVA: *p<0.05, **p<0.01.</p

Targeted Disruption of <i>LDLR</i> Causes Hypercholesterolemia and Atherosclerosis in Yucatan Miniature Pigs

<div><p>Recent progress in engineering the genomes of large animals has spurred increased interest in developing better animal models for diseases where current options are inadequate. Here, we report the creation of Yucatan miniature pigs with targeted disruptions of the low-density lipoprotein receptor (<i>LDLR</i>) gene in an effort to provide an improved large animal model of familial hypercholesterolemia and atherosclerosis. Yucatan miniature pigs are well established as translational research models because of similarities to humans in physiology, anatomy, genetics, and size. Using recombinant adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer, male and female <i>LDLR+/−</i> pigs were generated. Subsequent breeding of heterozygotes produced <i>LDLR−/−</i> pigs. When fed a standard swine diet (low fat, no cholesterol), <i>LDLR+/−</i> pigs exhibited a moderate, but consistent increase in total and LDL cholesterol, while <i>LDLR−/−</i> pigs had considerably elevated levels. This severe hypercholesterolemia in homozygote animals resulted in atherosclerotic lesions in the coronary arteries and abdominal aorta that resemble human atherosclerosis. These phenotypes were more severe and developed over a shorter time when fed a diet containing natural sources of fat and cholesterol. <i>LDLR</i>-targeted Yucatan miniature pigs offer several advantages over existing large animal models including size, consistency, availability, and versatility. This new model of cardiovascular disease could be an important resource for developing and testing novel detection and treatment strategies for coronary and aortic atherosclerosis and its complications.</p></div

Abdominal Aortic Atherosclerosis from 11-month old <i>LDLR+/+, LDLR+/−</i>, and <i>LDLR−/−</i> pigs fed a high fat, high cholesterol diet.

<p>All values are presented with SD. Differences between <i>LDLR−/−</i> and the other two genotypes are significant where indicated, ANOVA: **p<0.01.</p

Abdominal aortic and coronary atherosclerosis in a 26-week-old <i>LDLR−/−</i> pig fed a standard diet.

<p>Measurements of percent surface area with raised lesion in the abdominal aorta were taken from en face photographs of the abdominal aorta and confirmed by Sudan IV staining (A). Aortic sections (rectangle) were stained with VVG and H&E (B,C), higher magnifications of atherosclerotic lesion (squares) are seen in D and E. A representative section (with corresponding higher magnification) from the circumflex artery showing atherosclerotic plaque that appears to have foam cells. (F,G–H&E H,I – VVG).</p

Summary of total cholesterol from <i>LDLR+/+</i>, <i>LDLR+/−</i>, and <i>LDLR−/−</i> pigs fed a high fat, high cholesterol diet for 90 and 180 days.

<p>All values are presented with SD. Differences between <i>LDLR+/+</i> and <i>LDLR+/−</i> are significant where indicated, ANOVA: *p<0.05, **p<0.01. Differences between <i>LDLR−/−</i> and the other two genotypes are significant where indicated, ANOVA: **p<0.01.</p

Summary of <i>LDLR</i> gene targeting and SCNT activity.

<p>*Gene targeting efficiency reported as percentage of G418^R clones that were properly targeted, as determined by PCR.</p>†<p>Pregnancy rate refers to full-term gestation.</p

Summary of lipid profiles from <i>LDLR+/+</i>, <i>LDLR+/−</i>, and <i>LDLR−/−</i> pigs at birth and after 26 weeks on a standard diet.

<p>All values are presented with SD. Differences between <i>LDLR+/+</i> and <i>LDLR+/−</i> are significant where indicated, ANOVA: *p<0.05, **p<0.01. Differences between <i>LDLR−/−</i> and the other two genotypes are significant where indicated, ANOVA: *p<0.01.</p

<i>LDLR</i>-targeted pigs.

<p>A. Gene targeting strategy. Wild-type <i>LDLR</i> exons 2–6 are shown as gray boxes. <i>Neo^R</i> cassette is depicted in red, with the arrow showing the orientation relative to <i>LDLR</i>. The neomycin resistance cDNA is driven by a <i>PGK</i> promoter and flanked by <i>loxP</i> sites. The engineered termination codon is indicated. Position of the <i>LDLR</i> Southern blot probe is shown, as are the <i>Xmn</i>I restriction sites. B. <i>LDLR+/−</i> piglets at 2 days of age. C. Southern blot of genomic DNA from <i>LDLR+/−</i> cloned pigs. (Upper) <i>XmnI</i> digested genomic DNA was hybridized with a probe that detects porcine <i>LDLR</i> downstream of the targeting vector boundary. The <i>LDLR</i>-targeted allele produced an approximately 7.8 kb band, and the wild-type band is approximately 6.0 kb. (Lower) The same DNA was hybridized with a probe that detects the <i>Neo^R</i> cassette, yielding only the targeted 7.8 kb band. Lanes 1–5 contain DNA from individual cloned <i>LDLR+/−</i> pigs; lane 6 contains DNA from a wild-type pig. D. Sequence chromatogram of the site of <i>LDLR</i> disruption by the <i>Neo^R</i> cassette. The engineered termination codon is noted. E. Total cholesterol levels in plasma from 15–18 month old <i>LDLR+/−</i> pigs (n = 13) and wild-type controls (n = 7), *p<0.0001. Data presented with SD.</p

Molecular and biochemical characterization of <i>LDLR+/+</i>, <i>LDLR+/−</i>, and <i>LDLR−/−</i> pigs.

<p>A. Genotyping by PCR. Expected sizes are 1.5-type <i>LDLR</i> and 3.2 kb for targeted <i>LDLR</i>. B. Southern blot of genomic DNA. (Left) <i>XmnI</i> digested genomic DNA was hybridized with a probe that detects porcine <i>LDLR</i> downstream of the targeting vector boundary. The <i>LDLR</i>-targeted allele produced an approximately 7.8 kb band, and the wild-type band is approximately 6.0 kb. (Right) The same DNA was hybridized with a probe that detects the <i>Neo^R</i> cassette, yielding only the targeted 7.8 kb band. C. Northern blot of <i>LDLR</i> and <i>GAPDH</i> mRNA. Full-length <i>LDLR</i> and <i>GAPDH</i> mRNAs are 5.1 and 1.5 kb, respectively. The asterisk represents a minor mRNA species consisting of full-length <i>LDLR</i> mRNA plus the <i>Neo^R</i> cassette. The bracket indicates two minor mRNA species that are likely the result of nonsense-mediated mRNA altered splicing. D. Representative RT-PCR. Using PCR primers that amplify from exon 1 to exon 5, the targeted <i>LDLR</i> allele produces no normal mRNA, but does produce mRNA species with deletions of exon 4 or exons 3 and 4. This is seen in both the <i>LDLR+/−</i> and <i>LDLR−/−</i> pigs. This result was confirmed by DNA sequencing. E. Representative western blot of LDLR and β-tubulin. LDLR is ∼150 kDa and β-tubulin is 51 kDa.</p