At weaning, F2 mice were group housed with ad libitum access to water and a standard chow diet (Purina Rodent Chow 5001, Dean’s Animal Feed) with approximately 12% energy from fat (Research Diets D12492, Research Diets, New Brunswick, NJ). One group of male B6.S-2 congenic mice were also fed a purified high fat diet with 60% energy from fat.
Extracted molecule
total RNA
Extraction protocol
Total RNA from whole brain, epididymal white adipose tissue, gastrocnemius skeletal muscle, and liver were isolated using TRIzol reagent (Gibco/BRl, Grand Island, NY) followed by further purification with QIAGEN RNeasy kit (QIAGEN Inc., Valencia, CA) using the RNA cleanup protocol. cRNA was then hybridized to a custom NimbleScreen 12-well array platform.
Label
biotin
Label protocol
cDNA was transcribed into cRNA using the MEGAscript T7 Kit (Ambion, Austin, TX) with biotin labeled CTP and UTP followed by purification with RNeasy (QIAGEN, Valencia, CA).
Hybridization protocol
cRNA was then hybridized according to the NimbleGen protocol
Scan protocol
Maskless Array Synthesizer (MAS) technology (Nuwaysir, Huang et al. 2002)
Description
We previously constructed a congenic mouse, B6.S-D2Mit194-D2Mit311 (B6.S-2) with SPRET/Ei donor DNA on distal chromosome 2 in a C57BL/6J background that captured an obesity quantitative trait locus (QTL). Mice homozygous for SPRET/Ei alleles at the donor region had decreased body weight and obesity related phenotypes. The B6.S-2 congenic donor region spanned a minimum of 26 Mb. In this study, we constructed five overlapping subcongenics with smaller SPRET/Ei donor regions to fine map the underlying gene(s). One of the five subcongenic lines derived from the B6.S-2 founding congenic, B6.S-2A, captures most of the body weight and adiposity phenotypes in a donor region with a maximum size of 7.4 Mb. Homozygous SPRET/Ei donor alleles in both the founding congenic and the derived B6.S-2A subcongenic exhibit significant decreases in body weight, multiple fat pad weights with the greatest decrease in mesenteric fat pad weight, and Adiposity Index (total fat pad weight divided by body weight). Interval specific microarray analysis in four tissues for donor region genes from the founding B6.S-2 congenic identified several differentially expressed genes mapping to the B6.S-2A subcongenic donor region, including prohormone convertase 2 (PC2). Quantitative real-time PCR confirmed a modest decrease of PC2 expression in whole brains of mice homozygous for SPRET/Ei donor alleles. Analysis of the relative levels of mRNA for B6 and SPRET/Ei in heterozygous congenic mice showed differentially higher expression of the C57BL/6J allele over the SPRET/Ei allele indicating a cis regulation of differential expression. Using subcongenic mapping, we have successfully narrowed a body weight and obesity QTL interval and identified PC2 as a positional candidate gene.
