HMDPpheno11 project protocol

Diet effects on aortic lesion size in 103 Hybrid Mouse Diversity Panel strains that are transgenic for human APOE*3Leiden and human CETP (F1 hybrids)   (2015)

Bennett BJ, Lusis AJ
With: Davis RC, Civelek M, Orozco L, Wu J, Qi H, Pan C, Packard RR, Eskin E, Yan M, Kirchgessner T, Wang Z, Li X, Gregory JC, Hazen SL, Gargalovic PS

Project protocol - Contents

Workflow and sampling

Step Description Equipment Data collected
1 Confirmation of transgene presence in F1 hybrids at 8 weeks of age. Genotyping
2 Test mice fed a high-fat, high-cholesterol diet for 16 weeks.
3 Mice euthanized and lesion area of proximal aorta determined. Dissection kit and microscope Aortic lesion area


Procedure 1: Genotyping of F1 progeny and administration of high-fat diet

Definitions & Abbreviations: APOE*3Leiden: apolipoprotein E3 Leiden variant; CETP: cholesterol ester transfer protein

Equipment, software, and supplies

  • Western Style synthetic high-fat diet, 33 kcal % fat from cocoa butter supplemented with 1% cholesterol (Research Diets Inc., D10042101, New Brunswick, NJ, USA)


  1. F1 hybrids are generated from the breeding of females from the Hybrid Mouse Diversity Panel and C57BL/6J-Tg(hAPOE*3Leiden,hCETP) males.
  2. At about 8 weeks of age, F1 progeny from parent crosses are genotyped for the presence of human ApoE*3 Leiden and human CETP transgenes.
  3. Transgenic mice are placed on a synthetic high-fat diet for 16 weeks.

Procedure 2: Tissue collection and aortic lesion area estimation

Equipment, software, and supplies

  • Dissection kit
  • Microscope


Introductory Comments: Estimation of aortic lesion area is described in Shih DM et al., 2000.

  1. At 23-25 weeks of age, mice are euthanized using deep anesthesia with isoflurane vapor followed by cervical dislocation.
  2. The upper portion of the heart and proximal aorta are obtained, flushed with PBS, and embedded in OCT compound, and stored at -70°C.
  3. Serial 10 µm cryosections of aorta, beginning at the aortic root, are collected for a distance of 400 µm. These sections are stained with Oil Red O and hematoxylin and lesion area is quantified in every third section through the proximal aorta.


Primary References

Bennett BJ, Davis RC, Civelek M, Orozco L, Wu J, Qi H, Pan C, Packard RR, Eskin E, Yan M, Kirchgessner T, Wang Z, Li X, Gregory JC, Hazen SL, Gargalovic PS, Lusis AJ. Genetic Architecture of Atherosclerosis in Mice: A Systems Genetics Analysis of Common Inbred Strains. PLoS Genet. 2015 Dec;11(12):e1005711. doi: 10.1371/journal.pgen.1005711. Epub 2015 Dec 22.   PubMed 26694027     FullText

Protocol References

Shih DM, Xia YR, Wang XP, Miller E, Castellani LW, Subbanagounder G, Cheroutre H, Faull KF, Berliner JA, Witztum JL, Lusis AJ. Combined serum paraoxonase knockout/apolipoprotein E knockout mice exhibit increased lipoprotein oxidation and atherosclerosis. J Biol Chem. 2000 Jun 9;275(23):17527-35.   PubMed 10748217