DGAP: Lusis 2006-04-24

Expression Profiling of Liver Tissue from C57BL6 x DBA F2 Mice

A. Jake Lusis, Ph.D.
Depts of Medicine, Microbiology, and Human Genetics
Center for Health Sciences
UCLA

Thomas A. Drake, M.D.
Dept of Pathology and Lab Medicine
Center for Health Sciences
UCLA

Data contributed by Leslie Ingram-Drake, April 24, 2006

Summary

Expression profiling of liver tissue from 111 F2 female mice resulting from an F2 intercross between C57BL/6J and DBA/2J inbred mouse strains. Mice were additionally studied for obesity, plasma lipid, and atherosclerosis related traits, and genotyped to allow for QTL analysis of phenotypic and expression traits. Phenotype, genotype and liver expression data are provided here.

Mice were fed a chow diet for 12 months followed by 4 months of an atherogenic high-fat, high-cholesterol, cholate containing diet (Diet # 90221; Harlan Teklad, Madison, WI). Parental and F2 mice were killed at 16 months of age. Body weight and length measurements were obtained and blood collected immediately prior to sacrifice. The heart and ascending aortae were removed en bloc and embedded in OCT compound and frozen at -70C for subsequent sectioning for atherosclerotic lesion quantitation. Kidneys and spleens were collected for DNA isolation and fat pads (omental, retroperitoneal, parametrial, and subcutaneous) were removed and weighed. The livers were removed immediately and flash frozen at stored at -80C before expression profiling. Blood was collected by retro-orbital bleeding under isoflurane anesthesia from mice fasted for fourteen hours. Samples were collected prior to placement of the mice on the HF diet (chow diet samples) and at time of sacrifice (high fat, or HF, diet samples). Plasma was separated from cells by centrifugation of blood at 12,000 x g for 3 min. Total cholesterol, HDL cholesterol, triglycerides, and unesterified free fatty acids were performed using established enzymatic assays. Plasma insulin and leptin were measured by immunoassay. Data for all these traits from each mouse are provided. Not all traits were able to be obtained on all mice. (See publications PMID: 11328966 and 12925895 for further details. The 111 mice included here are a subset on which expression studies were obtained).

Genotyping was performed by PCR amplification of microsatellite markers spaced at an average of 13 cM. PCR products were run on 5% polyacrylamide gels and detected by autoradiography. Genotypes at each marker are designated as B (homozygous C57BL/6J), D (homozygous DBA/2J), or H (heterozygous). Comprehensive microarray analyses were performed using a curated set representing 23,574 murine genes and ESTs, and 2,186 control sequences. Arrays were manufactured by Agilent Technologies, and used 60mer oligonucleotide probes constructed on the arrays using ink-jet technology. Total RNA was extracted using Triazol reagent. For these F2 intercross samples, control RNA is composed of a pool derived from all F2 and parental samples. RNA quality was validated prior to analysis. Portions were reverse transcribed and labeled with either Cy3 fluorochrome or alternatively with Cy5 fluorochrome. Each labeled cDNA was hybridized along with its alternatively-labeled control cDNA on duplicate arrays, with the labeling of sample and control reversed between the two arrays. Array images were scanned using an Agilent Dual Laser Microarray scanner. Data were processed to obtain background noise, single-channel intensities, and associated measurement error estimates, and intensity values were accordingly normalized and background-corrected. Measurements derived were expressed as mean log10 intensity (MI) and mean log10 ratio (MR) of sample to control values, derived from the duplicate reverse-fluor hybridizations. An error model for the MR value was applied to quantify the significance of expression change between sample and control. (See publication PMID: 12646919, and GEO record GDS923 for further details).

Data Files

a_substance_id - Rosetta generated unique probe identifier
mlratio - MLRATIO: Averaged log10 ratio [log10(CHANNEL1/CHANNEL2), error weighted]
mlaverage - Average log intensity of channels
pvalue - P-value of LogRatio
mlratioerr - Error estimate for MLRATIO (VALUE)

a_substance_id - Rosetta generated unique probe identifier
SEQUENCE_ID - Identifier of sequence associated with the gene represented on the microarray
GENE_SYMBOL - Gene symbol from LocusLink
GB_ACC - GenBank accession number associated with the gene represented on the microarray
SEQUENCE - Sequence of oligonucleotide probe

Diabetes Genome Anatomy Project

Expression Profiling of Liver Tissue from C57BL6 x DBA F2 Mice

Summary

Data Files