MOUSE :: HMDP :: Original panel

A.         Hybrid Mouse Diversity Panel:  Original Panel Analysis

1.         Mice (B. Bennett)
2.         Plasma lipids, insulin & glucose assays (L. Castellani)
3.         Tissues, weights (B. Bennett)
4.         Bone density (Charles Farber)
5.         Liver gene expression arrays (B. Bennett)
6.         Adipose gene expression arrays (B. Bennett)
7.         Bone gene expression arrays (Charles Farber)
8.         Blood cell parameters (B. Bennett)
9.         Liver metabolites, Duke U. (B. Bennett)
10.        Brain, striatum, expression arrays (D. Smith)
11.        Brain, cerebellum, expression arrays (D. Smith)
12.        Liver proteomics (A. Ghazalpour)
13.        Body composition, NMR (B. Bennett)
14.        Association analysis (B. Bennett)
15.        Behavioral traits (D. Smith)
16.        HDL parameters (D. Shih)

 

1.         Mice (B. Bennett)

Male mice from the hybrid HMDP panel were purchased from the Jackson Labs. Mice were between 6 and 10 wk of age and to ensure adequate acclimatization to a common environment the mice were aged until 16 wk of age. All mice were maintained on a chow diet (Ralston-Purina Co.) until sacrifice at 16 wk of age. A complete list of strains included in the study is listed in Supplemental Table 1. Fol- lowing a 16-h fast, mice were bled retro-orbitally under isoflurane anesthesia. Plasma lipids were determined as previously described (Mehrabian et al. 1993). Mice were euthanized by cervical disloca- tion, livers dissected out and flash frozen in liquid nitrogen. Genotyping Inbred strains were previously genotyped by the Broad Institute (http://www.broadinstitute.org/mouse/hapmap), and they are com- bined with the genotypes from Wellcome Trust Center for Human Genetics (WTCHG). Genotypes of RI strains at the Broad SNPs were inferred from WTCHG genotypes by interpolating alleles at poly- morphic SNPs among parental strains, calling ambiguous geno- types missing. Details of genotype imputation are in Supplemental Methods. Of the 140,000 SNPs available, 107,145 were informative with an allele frequency greater than 5% and were used for GWAS.

Inbred and RI strains used in mouse whole genome association can be found in the following table:

hmdp_mice_table

 

2.         Plasma lipids, insulin & glucose assays (L. Castellani)

General Overview: The assays that we routinely perform are total cholesterol, HDL cholesterol, unesterified cholesterol (UC), unesterified (free) fatty acids (FFA), triglycerides (TG) and glucose. Total plasma cholesterol is assayed after treating the samples with cholesterol esterase to hydrolyze cholesterol esters and then performing the cholesterol assay, which will now encompass all of the cholesterol that was in both the cholesterol ester and unesterfied cholesterol pools. Hence, this is the “total” cholesterol assay. Unesterified cholesterol is assayed by performing the cholesterol assay without treating the plasma sample with cholesterol esterase, so that only the free or unesterified cholesterol is determined. This value can then be subtracted from the total cholesterol to calculate the esterfied or cholesterol esters (CE). The HDL cholesterol is determined by performing the total cholesterol assay on the supernatant after precipitation of the apoB containing lipoproteins with Heparin/Manganese Chloride. The triglyceride assay is a typical assay that actually determines the mass of glycerol released after the hydrolysis of fatty acids from triglycerides. However, we also do a triglyceride (glycerol) “blank” assay to measure the endogenous levels of glycerol present in the mouse plasma prior to hydrolysis of triglycerides. This endogenous “blank” value is then subtracted from the total glycerol determined after the hydrolysis of triglycerides in order to correct for the endogenous plasma free glycerol concentrations, which are considerably higher in mice than in humans. Most of the pipetting is performed using a Beckman Biomek 2000 Automated Workstation with the assays read in a 96 well format using a Molecular Devices Spectramax-Plus microplate reader.

Quality Control:
Internal quality control: All assays are run in triplicate determinations on a 96 well plate. We also run standards on each plate as well as control samples with known analyte concentrations on each plate in order to validate the accuracy of the assay. This allows us to run 26 unknown samples (in triplicate) on each plate, with the remainder of the wells used by the standards, control samples, and sample blanks, all of which are also run in triplicate.

External quality control: We participate in the Centers for Disease Control and Prevention Lipid Standarization program. Our laboratory ID # is LSP-251. Each quarter we receive 12 test samples from the CDC, and one of the CDC test samples is included with an actual run of our unknown samples each week. The values we obtain from the test samples are submitted to the CDC at the end of each quarter, and we are notified if we have met their criteria for accuracy and precision. We have passed each quarter for the past 18 years.

Supplies/Reagents needed:

A.  0.9 % sodium chloride solution
B.  0.65ml minitubes (Phenix catalog# M-931B)
C. 96 well round bottom plates (USA Scientific catalog #5665-0101)
D.  96 well flat bottom plates (USA Scientific catalog #5665-    5101)
E.   Reagents for triglyceride and triglyceride blank assays

Triglyceride Assay Reagents: Triglyceride reagent (Sigma catalog # F6428 triglyceride [free glycerol] reagent) reconstituted as per manufacturers recommendations and 8000U lipase (EMD Biosciences catalog #437707). Reagent for triglyceride ‘blank’ assay is made exactly the same way but the lipase is omitted.

F.  Glucose assay reagent (Fisher catalog # SB1070-125)
G.  Glucose standard (Sigma catalog # G6152)
H.  Triglyceride standard (Sigma catalog #G556-100ml)
I.    Cholesterol standard (Fisher catalog # SB1012-030)
J.  FFA assay reagents (reconstituted per manufacturers recommendations)
NEFA Color reagent A (Wako catalog# 999-34691)
NEFA Solvent A: (Wako catalog# 995-34791)
NEFA Color reagent B: (Wako catalog# 991-34891)
NEFA Solvent B: (Wako catalog# 995-35191)
NEFA Standard solution (1mEq/L) (Wako catalog # 276-76491)

K.  Control samples
Control 1- SER-T-FY-1 Level 1 human control serum (Stanbio cat # G427-86)
Control 2 is SER-T-FY-2 Level 2 human control serum (Stanbio cat # G428-86).
L.  Reagents for cholesterol assays

Cholesterol Assay Reagent:
4-amino antipyrine (Aldrich catalog # A3,930-0), KCl (Fisher catalog # P217-500), sulfonic acid (Research Organics catalog # 6062H-3), sodium cholate (USB catalog # 13630), pipes (Sigma catalog # P-3768), Triton X-100 (Sigma catalog # T-6878), horseradish peroxidase (Amresco catalog # 0417), cholesterol oxidase (EMD Biosciences catalog # 228250) and cholesterol esterase (EMD Biosciences catalog # 228180) [Omit the cholesterol esterase from the reagent for ‘UC’ assay]

Heparin/MnCl2 for precipitation reagents for HDL assay     
MnCl2 (Fisher catalog # M87-100), Heparin solution (EMD Biosciences catalog # 375095)

Preparation of Plasma Samples, Standards, Controls, and Blanks:

General Overview: The enzymatic colorimetric assays are read on a 96 well plate in a Molecular Devices SpectraMax plus plate reader. This allows the analysis of 26 samples per plate when run in triplicate determinations, with the remainder of the wells used for the 3 standards, 2 control plasmas, and 1 assay blank, all of which are also run in triplicate. Therefore, a total of 32 minitubes should be labeled for each assay being run (26 for the samples, 3 for standards, 2 for control plasmas and 1 for the assay “blank”). After the tubes are labeled proceed with the preparation of the plasma samples, standards, controls and blanks for each assay as described below.

Initial Dilution of Plasma Samples: The frozen mouse plasma samples, which should have at least 100ul per tube, are thawed on ice. Once thawed they are vortexed and centrifuged in a table top microcentrifuge to recover all of the sample at the bottom of the tube. Depending on the strains and diets, the plasma is diluted from 4 to 16 fold with 0.9% NaCl, since very lipemic samples can occur with genetically modified mouse strains on a high fat/high cholesterol diet. For assays of common inbred strains on a normal low fat chow diet, we take 75 ul of plasma and add 225 ul of 0.9% saline for a 4 fold dilution. This will give enough total diluted sample to run all of the assays in triplicate. Obviously, for lipemic samples that will be diluted more, a smaller volume of plasma is sufficient to run all assays. The goal of the dilutions is have the lipid concentrations high enough to give reliable OD readings significantly above background, without exceeding or approaching the maximal OD reading of the plate reader. Thus, different batches of samples have to be diluted differently to meet these requirements.

Preparing Standards for each assay: Standards for the various assays are initially prepared at the concentrations listed below for each assay. Each standard is then diluted with 0.9% NaCl to the same fold dilution as the unknown plasma samples, with the following exceptions. The most concentrated cholesterol standard (standard 4; 400 mg/dl) is diluted only half as much as the unknown plasma samples, and none of the FFA standards undergo further dilution after they are prepared.

Triglyceride (glycerol) standards: Standard 1- 4.81 mg/dl glycerol (equivalent to 46.25mg/dl TG), Std 2- 9.62 mg/dl glycerol (equivalent to 92.5mg/dl TG), Standard 3- 19.24 mg/dl glycerol (equivalent to 185mg/dl TG) and Standard 4- 38.48 mg/dl glycerol (equivalent to 370mg/dl TG).

Cholesterol Standards: Standards 5 and 4 are prepared by taking the cholesterol standard (Fisher catalog # SB1012-030; 200mg/dl) directly as provided. Standards 3 through 1 are then prepared by serial twofold dilutions from standard 4. Thus, the actual initial concentrations of your standards are Standard 1- 25 mg/dl, Standard 2- 50 mg/dl, Standard 3- 100 mg/dl, Standard 4- 200 mg/dl, and Standard 5- also 200 mg/dl. When performing the assay, standards 4 through 1 are diluted with 0.9% NaCl to the same fold dilution as the unknown samples, while Std 5 undergoes only half the dilution of the unknowns.

FFA (Nonesterified fatty acid) standards: The FFA concentration in the standard solution we order (Wako catalog # 276-76491) is 28.25 mg/dl. Standard 1 is the stock solution diluted 1.5 x of your sample dilution and is labeled 18.83 mg/dl. Standard 2 is the stock at the same dilution as your samples and is labeled 28.25 mg/dl. Standard 3 is the stock diluted 0.5x your sample dilution and is labeled 56.5 mg/dl.

Glucose standards: The glucose (Sigma catalog #G6152) standards are made at the following conentrations. Standard 1; 100 mg/dl, Standard 2; 200 mg/dl, and standard 3; 400 mg/dl. Once the three standards have been prepared, they are each then diluted further with 0.9% NaCl to the same extent as the unknown samples.

Preparing Control Samples: We run two control samples, a low value (Control 1) and a high value (Control 2), for each assay on each 96-well plate. The controls are purchased from Stanbio (Boerne, Tx, USA). Control 1 is SER-T-FY-1 Level 1 human control serum (cat # G427-86) and Control 2 is SER-T-FY-2 Level 2 human control serum (cat # G428-86). The exact concentration of each analyte varies slightly by lot# and the specific values for a lot are included on the lot specification sheet with each shipment. The values for each analyte for each control are generally in the following ranges:
Glucose- Control 1; 95mg/dl  Control 2; 300mg/dl
Cholesterol- Control 1; 95mg/dl  Control 2; 300mg/dl
Free fatty acids- Control 1; 9mg/dl  Control 2; 40mg/dl
HDL chol- Control 1; 95mg/dl  Control 2; 300mg/dl

Preparing Assay blanks: Sample “blanks” are prepared for each assay by adding 75 ul of saline, rather than plasma, to make the initial dilutions from which aliquots are taken for all of the assays. This “blank” is then run exactly the same way as the unknown plasma samples for all of the assays (except HDL, see below), and the “blank” OD reading (which should be essentially zero) is subtracted from all other values. In addition to the saline “blank” described above, the HDL assay also includes a heparin-MnCl2 blank, since there is a heparin-MnCl2 precipitation step in the HDL assay. In the case of the heparin-MnCl2 blank, you do not actually have a precipitate (since it has saline instead of plasma), but take 30uls of the “supernatant” just as you would for the samples which contain plasma. This “blank” from the precipitation is used to subtract from the HDL cholesterol values obtained for the unknown and control samples, while the saline “blank”, prepared as described above, is used for the standards on the HDL assay.

Setting up for the Individual Assays:

Total cholesterol, unesterified cholesterol, and HDL cholesterol assays:

Additional Step for HDL Assay Only: The cholesterol assays for total cholesterol and unesterified cholesterol are done directly on aliquots of the diluted plasma samples. For the analysis of HDL cholesterol, prior to running the cholesterol assay the HDL has to be isolated from the other lipoproteins by precipitation. The apoB containing lipoproteins are precipitated from 100ul of the diluted plasma in the 96 well U-bottom plates using heparin-MnCl2. The plates are then centrifuged for 30min at 40c at 2500rpm, in a Beckman TJ-6 (or comparable) centrifuge.  30ul of the supernatants are then taken for the cholesterol assay. Since the plasma sample is diluted further in the heparin-MnCl2 precipitation step, the HDL cholesterol values have to be multiplied by 1.2.

Cholesterol Assay: The total cholesterol assay is done on 20ul of the diluted plasma sample, the UC assay on 25ul of the diluted plasma sample, and the HDL assay on 30ul of the supernatant after heparin-MnCl2 precipitation. The samples, controls and standards are added to 0.65 ml minitubes to which 600 ul of the cholesterol reagent is added. The reagent with esterase is used for the total cholesterol and HDL assays, and the reagent without esterase is used for the UC assay. The samples are then incubated at 37degrees C in a water bath. After the incubation 170ul aliquots in triplicates are loaded into the 96 well flat bottom plates. Read the plates at 515nm, subtracting the “blank” values from all readings.. Because the total cholesterol is higher than unesterified or HDL cholesterol, use standards 100 mg/dl, 200 mg/dl, and 400 mg/dl. For the HDL assays, use standards 50 mg/dl, 100 mg/dl, and 200 mg/dl. For the UC assay use standards 25 mg/dl, 50 mg/dl, and 100 mg/dl.

Triglyceride and Triglyceride blank assays: For the triglyceride and triglyceride blank assays aliquot 30ul of the diluted sample, controls, and standards into 0.65ml minitubes. Use Glycerol standards 46.25 mg/dl, 92.5 mg/dl, and 185 mg/dl for the triglyceride blank assay and glycerol standards 92.5 mg/dl, 185 mg/dl, and 370 mg/dl for the triglyceride assay. Add 600uls of the triglyceride assay reagent with lipase to each tube for the triglyceride assays, and add 600uls of the triglyceride reagent without lipase to each tube for the triglycride blank assay. Incubate the tubes for 10 min at 37 degrees C in a water bath.  After the incubation load 170ul in triplicates into 96 well flat bottom plates. Read the plates at 540nm. After running the triglyceride and triglyceride blank assays for each sample, the value of the triglyceride blank is subtracted from the triglyceride value to correct for endogenous levels of free glycerol in the plasma.

FFA assay: For the free fatty acid assay aliquot 30ul of the diluted plasma sample, controls, and standards into 0.65ml minitubes. Add 400uls of reagent A and incubate for 5 min at 37 degrees C in a water bath. Then add 200uls of reagent B and incubate for 5 min at 37 degrees C in a water bath.  After incubation load 170ul in triplicates into 96 well flat bottom plates. Read the plates at 550nm and subtract “blank” values from the reading.

Glucose assay:
For the glucose assay, aliquot 15ul of the diluted plasma, controls, and standards into 0.65ml minitubes. Add 600uls of the glucose reagent directly as supplied by the manufacturer, (Fisher cat # SB1070-125 manufactured by Stanbio) to each tube and incubate for 5 min at 37 degrees C in a water bath. After the incubation load 170ul in triplicates into 96 well flat bottom plates. Read the plates at 505nm. Subtract the assay “blank” from the values.

Insulin assay:
Insulin levels in plasma were measured using the ALPCO Mouse Ultrasensitive Insulin ELISA according to manufacturer’s instructions as described : http://www.alpco.com/products/Insulin_Ultrasensitive_Mouse_ELISA.aspx

 

3.         Tissues, weights (B. Bennett)

Weight of following tissues taken: gonadal, femoral, mesenteric fat pads, heart and on a subset of the
NMR analysis.

 

4.         Bone density (Charles Farber)

BMD determination: All carcasses were stored at -20°C after sacrifice and then thawed overnight at 4°C prior to BMD scans.  The entire thawed carcass was scanned.  BMD scans were performed using a Lunar PIXImus II Densitometer (GE Healthcare, Piscataway, NJ).  The PIXImus II was calibrated daily using a phantom of known BMD.  BMD was calculated for the entire carcass minus the skull, the lumbar spine and the left femur. 


1.         Farber CR, Bennett BJ, Orozco L, Zou W, Lira A, et al. (2011) Mouse genome-wide association and systems genetics identify Asxl2 as a regulator of bone mineral density and osteoclastogenesis. PLoS Genet 7: e1002038. doi:10.1371/journal.pgen.1002038

 

5.         Liver gene expression arrays (B. Bennett)

RNA isolation and Expression Profiling: Initial profiling studies were performed on liver tissue.  Flash frozen samples were weighed and homogenized in Qiazol according to the manufacturer’s protocol.  Following homogenization livers were isolated in RNeasy 96 columns (Qiagen) using the manufacturers protocol.  RNA integrity was confirmed using the Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA).

Microarray Sample Preparation, Randomization: RNA was isolated from liver samples  from the 99 strains comprising the HMDP. 92 strains of mice had three biological replicates, five strains had two biological replicates and two strains with one biological replicate each. All RNA samples were cleaned using a Biosprint96 (Qiagen, Valencia, CA) with RNA cleanup beads (Agencourt Bioscience, Beverly, MA) following manufacturer’s protocol with adaptations for use with the Biosprint. The quality of the total RNA from the those samples were monitored by the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) and RNA quantity was measured with a NanoDrop (NanoDrop Technologies, Inc. Wilmington, DE) following the manufacturer's instructions. All samples were arrayed into three 96 well microtiter plates following a randomized design format that places samples from the same strain on different plates to better estimate variance across testing strains.

Microarray Target Labeling Hybridization, and Quality Control: All target labeling reagents were purchased from Affymetrix (Santa Clara, CA). Double-stranded cDNAs were synthesized from 1ug total RNA through reverse transcription with an oligo-dT primer containing the T7 RNA polymerase promoter and double strand conversion using the cDNA Synthesis System. Biotin-labeled cRNA was generated from the cDNA and used to probe Affymetrix Mouse Genome HT_MG-430A arrays. The HT_MG-430A Array plate consists of 96 single MG-430A arrays arranged into standard SBS 96 well plate format.  All cDNA and cRNA target preparation steps were processed on a Caliper GeneChip Array Station from Affymetrix. Array hybridization, washing and scanning were performed according to the manufacturer’s recommendations.  Scanned images were subjected to visual inspection and a chip quality report was generated by the Affymetrix's GeneChip Operating System (GCOS) and Expression console (Affymetrix). Two of 288 chips were excluded due to low QC scores.  The image data was processed using the Affymetrix GCOS algorithm utilizing quantile normalization or the Robust Multiarray method (RMA) to determine the specific hybridizing signal for each gene. Expression data can be obtained from Geo databases for liver (GSE16780).

 

6.         Adipose gene expression arrays (B. Bennett)

This experiment was performed on the HMDP :: second set.

 

7.         Bone gene expression arrays (Charles Farber)

RNA and microarray processing: At sacrifice, the diaphysis of the right femur was excised and cleaned free of soft tissue.  Bone marrow was removed by flushing with PBS using a 22-guage needle and 3 ml syringe.  The bone was then flash frozen in LN2 and stored at -80C.  Total RNA was isolated using the Trizol Plus RNA Purification Kit (Invitrogen, Carlsbad, CA) following homogenization of the whole bone sample.  RNA integrity was confirmed using the Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA).  Microarray expression profiles were generated (N=1-3 per strain) using the Illumina MouseWG-6 v1.1 BeadChips (Illumina, San Diego, CA) by the Southern Genotyping Consortium at UCLA.  Biotin-labeled cRNA was synthesized by the total prep RNA amplification kit from Ambion (Austin, TX). cRNA was quantified and normalized to 77 ng/μl, and then 850 ng was hybridized to Beadchips.


Farber CR, Bennett BJ, Orozco L, Zou W, Lira A, et al. (2011) Mouse genome-wide association and systems genetics identify Asxl2 as a regulator of bone mineral density and osteoclastogenesis. PLoS Genet 7: e1002038. doi:10.1371/journal.pgen.1002038.

 

8.         Blood cell parameters (B. Bennett)

This experiment was performed on the HMDP :: second set.

 

9.         Liver metabolites (B. Bennett, Chris Newgard, Duke University)

Liver Metabolite Quantification: Amino acids, acyl-carnitines and organic acids were measured using stable isotope dilution techniques. Amino acids and acyl-carnitine species were measured using flow injection tandem mass spectrometry and sample preparation methods described previously. Briefly, samples were equilibrated with a cocktail of internal standards, de-proteinated by precipitation with methanol, aliquoted supernatants were dried, and then esterified with hot, acidic methanol (acyl-carnitines) or n-butanol (amino acids). The data were acquired using a Micromass Quattro micro TM system equipped with a model 2777 autosampler, a model 1525 µ HPLC solvent delivery system and a data system controlled by MassLynx 4.0 operating system (Waters, Milford, MA). Organic acids were quantified using a previously described method that utilizes Trace GC Ultra coupled to a Trace DSQ MS operating under Excalibur 1.4 (Thermo Fisher Scientific, Austin, TX).

Sixty-seven liver metabolites were measured, comprised of 15 amino acids and urea cycle intermediates, 45 acyl-carnitine derivatives, and 7 organic acids (TCA cycle intermediates and related analytes). The specific metabolites are listed in Table XXX. All MS analyses employed stable-isotope-dilution. The standards serve both to help identify each of the analyte peaks and provide the reference for quantifying their levels. Quantification was facilitated by addition of mixtures of known quantities of stable-isotope internal standards from Isotec (St. Louis, MO), Cambridge Isotope Laboratories (Andover, MA), and CDN Isotopes (Pointe-Claire, Quebec, CN) to samples, as follows: Acyl-carnitine assays–D3-acetyl, D3-propionyl, D3-butyryl, D9-isovaleryl, D3-octanoyl, and D3-palmitoyl carnitines; Amino acid assays–15N1,13C1-glycine, D4-alanine, D8-valine, D7-proline, D3-serine, D3-leucine, D3-methionine, D5-phenylalanine, D4-tyrosine, D3-aspartate, D3-glutamate, D2-ornithine, D2-citrulline, and D5-arginine; Organic acid assays–D3-lactate, D3-pyruvate, 13C4-succinate, D2-fumarate, D4-glutarate, 13C1-malate, D6-alpha-ketoglutarate, and D3-citrate. In addition to mass, analytes are identified on the basis of the particular MS/MS transitions that we monitor for each class of metabolites. For example, all acyl-carnitine methyl esters produce a fragment m/z 99. We make the assumption that all even mass precursors ions of m/z 99 are acyl-carnitines to which we assign plausible molecular structures. We differentiate isobaric struc ures e.g., dicarboxylic and hydroxylated acyl-carnitines, by comparing of MS/MS spectra for precursors of m/z 85 butylated acyl-carnitine species. We can infer whether the original compound had one or two carboxyl groups on the basis of the mass change from methyl to butyl esters.

 

10.       Brain, striatum, expression arrays (D. Smith)

Tissue harvesting: Brains were removed from each animal after euthanasia.  Hippocampi were dissected out and flash frozen in liquid nitrogen.  RNA was extracted from each sample using the Qiagen RNeasy kit.

Microarray data collection: Gene expression levels were quantified using Illumina Mouse-Ref 8 v2.0 Expression BeadChip microarrays.  The data were normalized using the rank invariant option in the software package BeadStudio (Illumina) (Kuhn et al., 2004). The microarray data are available at the Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) under accession number: GSE26500.

Kuhn K, Baker SC, Chudin E, Lieu MH, Oeser S, Bennett H, Rigault P, Barker D, McDaniel TK, Chee MS: A novel, high-performance random array platform for quantitative gene expression profiling. Genome Res 14:2347-2356, 2004.

Park CC, Gale GD, DeJong S, Ghazalpour A, Bennett B, Farber CR, Langfelder P, Lin A, Khan AH, Eskin E, Horvath S, Lusis AJ, Ophoff R, Smith DJ. Gene networks associated with conditional fear in mice identified using a systems genetics approach. BMC Syst Biol, 5:43, 2011.

 

11.       Brain, cerebellum, expression arrays (D. Smith)

Tissue harvesting: Brains were removed from each animal after euthanasia.  Striata were dissected out and flash frozen in liquid nitrogen.  RNA was extracted from each sample using the Qiagen RNeasy kit.

Microarray data collection: Gene expression levels were quantified using Illumina Mouse-Ref 8 v2.0 Expression BeadChip microarrays.  The data were normalized using the rank invariant option in the software package BeadStudio (Illumina) (Kuhn et al., 2004). The microarray data are available at the Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE26500.


Kuhn K, Baker SC, Chudin E, Lieu MH, Oeser S, Bennett H, Rigault P, Barker D, McDaniel TK, Chee MS: A novel, high-performance random array platform for quantitative gene expression profiling. Genome Res 14:2347-2356, 2004.

Park CC, Gale GD, DeJong S, Ghazalpour A, Bennett B, Farber CR, Langfelder P, Lin A, Khan AH, Eskin E, Horvath S, Lusis AJ, Ophoff R, Smith DJ. Gene networks associated with conditional fear in mice identified using a systems genetics approach. BMC Syst Biol, 5:43, 2011.

 

12.       Liver proteomics (A. Ghazalpour)

The protocol for this study can be found in the following reference:

Ghazalpour A, Bennett B, Petyuk VA, Orozco L, Hagopian R, Mungrue IN, Farber CR, Sinsheimer J, Kang HM, Furlotte N, Park CC, Wen PZ, Brewer H, Weitz K, Camp DG 2nd, Pan C, Yordanova R, Neuhaus I, Tilford C, Siemers N, Gargalovic P, Eskin E, Kirchgessner T, Smith DJ, Smith RD, Lusis AJ. Comparative analysis of proteome and transcriptome variation in mouse. PLoS Genet. 2011 Jun;7(6):e1001393. Epub 2011 Jun 9. PubMed PMID: 21695224.

 

13.       Body composition, NMR (B. Bennett)

Animals were measured for total body fat mass and lean mass by nuclear magnetic resonance (NMR) using the Bruker Minispec with software from Echo Medical Systems (Houston, TX).  All F2 mice were measured at 16 weeks of age on the day prior to euthanasia.

 

14.       Association analysis (B. Bennett)

Genome-wide association mapping accounting for population structure: We applied the following linear mixed model to account for the population structure and genetic relatedness among strains in the genome-wide association mapping. (Kang, Zaitlen et al. 2008)
y=μ+xβ+u+e
where μ represents mean, x represents SNP effect, u represents random effects due to genetic relatedness with Var (u) = σg2K and Var (e) = σe2, where K represents IBS (identity-by-state) matrix across all genotypes. A restricted maximum likelihood (REML) estimate of σg2 and σe2 are computed using EMMA (Efficient Mixed Model Association), and the association mapping is performed based on the estimated variance component with a standard F test to test β≠0. We defined an eQTL as local if the peak association signal was within a 10Mb sliding window of the physical location of the gene(s). We then calculated the average distance between these local eQTL and the transcription start site of the corresponding gene(s) transcription start site.

Estimation of power and mapping resolution: We evaluated the statistical power of the HMDP through simulation studies, with various parameters including the variance explained by SNP, variance explained by genetic background, and variance explained by random errors, and the number of repeated measurements per strain. For the comparison of power with single RI set or classical inbred (CI) only studies, we selected a subset of the simulated phenotypes for each RI or CI set and evaluated the power in the same way. Since there are 8 possibilities of SNPs being polymorphic among three sets of RI strains, the putative causal SNPs are categorized into 8 classes and power is evaluated for each class. The significance threshold per each RI set is determined separately using parametric bootstrapping described below.  See Supplemental Methods for comparison of BXD RI set to the full HMDP and simulations.

Genome-wide Significance Threshold: Genome-wide significance threshold in genome-wide association mapping is determined by the family-wise error rate (FWER) as the probability of observing one or more false positives across all SNPs per phenotype. We ran 100 different sets of permutation tests and parametric bootstrapping of size 1000, and observed that the mean and standard error of the genome-wide significance threshold at family-wise error rate (FWER) of 0.05 were 3.9 x 10-6 ± 0.3 x 10-6, and 4.0 x 10-6 ± 0.3 x 10-6, respectively. This is approximately an order of magnitude larger than the significance threshold obtained by Bonferroni correction (4.6 x 10-7). We also performed parametric bootstrapping under simulated the genetic background effect from population structure using EMMA. With 50% and 100% of variance explained by genetic background, the thresholds were determined to be 1.6 x 10-6 ± 0.2 x10-6 and 1.7 x 10-6 ± 0.2 x 10-6. The reduction in the significance threshold compared to no genetic background effect is due to the fact that inter-SNP correlation due to long-range LD reduces when conditioning on the population structure.  A detailed explanation of these analyses is provided in the Supplemental Methods.

 

15.       Behavioral traits (D. Smith)

Fear conditioning: All HMDP strains were exposed to a fear conditioning procedure followed by two independent memory tests. On each test day, mice were wheeled to a holding room for a 30 min acclimation period prior to testing.  Each mouse was tested individually and then transferred to a holding cage.  On day 1,mice were placed in a 25 cm x 20 cm conditioning chamber with grid floors and white plexiglass.  Following a 3 minute exploration period, mice received three auditory conditional stimuli (CS; 2000Hz, 15 seconds, 80 dB) co-terminating with footshock unconditional stimulus (US; 0.75 mA, 1 second), delivered with an inter-trial interval (ITI) of 1 minute.  Mice were removed 2 minutes following the final US.  On day 2, contextual fear was assessed.  Mice were then returned to the conditioning chamber under conditions identical to day 1.  Neither the CS nor US was presented during an 8 minute test.  On day 3, cued fear was assessed following a contextual shift.  Mice were placed in a novel, rectangular activity chamber (50 cm x 25 cm), given a 3 minute exploration period followed by a series of ten CS presentations (ITI 1 min), then removed from the chamber 1 minute following the final CS.  No US were presented during this test.  This apparatus was cleaned with 70% ethanol between tests.

Behavioral data analysis: Behavior was recorded digitally from a camera mounted above each test chamber, then digitized at 15 frames per second with the EthoVision Pro tracking system (Noldus Information Technology).  For each mouse a total of 48 unique endpoints were quantified automatically with EthoVision software (Table).  Varying numbers of biological replicates were obtained for each strain (ranging from n = 3 to n = 16, mean = 7.3).  These measures were designed to characterize multiple dimensions of defensive behavior.

Mean performance for each endpoint was determined by either collapsing across the entire test session for context fear endpoints or across specific test phases for fear conditioning (pre-US, post-US) and cued fear test (pre-CS, CS) endpoints.  The pre-US period consisted of the 3 minutes prior to the initial CS presentation, while the post-US period encompassed the 4.25 minute interval between the first US presentation and removal from the chamber.  Likewise, the pre-CS period spanned the 3 minutes prior to CS presentation, and the CS period covered the 12.5 minute period between the first CS presentation and removal from the chamber.  Measures reflecting rate changes were quantified by analyzing time course data within individual test phases. 

For the context test, endpoint rate changes were calculated as the percent change from the initial 2 minute epoch to the final 2 minute epoch.  For multi-phase tests (training, cued fear test), rate changes were calculated as suppression ratios based on mean values from the relevant test phases (pre/(pre+post)).  Strain means were calculated and served as the behavioral phenotypes for downstream analysis.  Velocity is the mean rate of movement in any given interval (e.g. cm/s), while mobility is the time spent mobile, expressed as a percentage of total time.

Classification of quantified behavioral phenotypes (48 total) can be found in the following table:

behaviorTable

 

Gale GD, Yazdi RD, Khan AH, Lusis AJ, Davis RC, Smith DJ. A genome-wide panel of congenic mice reveals widespread epistasis of behavior quantitative trait loci. Mol Psychiatry, 14:631-645, 2009.

Park CC, Gale GD, DeJong S, Ghazalpour A, Bennett B, Farber CR, Langfelder P, Lin A, Khan AH, Eskin E, Horvath S, Lusis AJ, Ophoff R, Smith DJ. Gene networks associated with conditional fear in mice identified using a systems genetics approach. BMC Syst Biol, 5:43, 2011.

 

16.       HDL parameters (D. Shih)

Cholesterol Efflux Study using 3H-Cholesterol with acetylated human LDL: Wears gloves during the entire procedure. RAW 264.7 cells, a mouse macrophage cell line, (300,000 cells/well) are plated in 24-well plates in DMEM (11965) + 10% FBS for 1 day.Cells are washed once with PBS and then incubated with 1 ml of DMEM with 10% FBS, 25 mg/ml human acetylated LDL, and 1 mCi 3H-Cholesterol/ml for 48 hours in a CO2 incubator. The specific activity of the original 3H-Cholesterol stock is 10-20 Ci/mmol. Ac-LDL stock: 2 mg/ml (80x). The media containing 3H-Cholesterol/ml are aspirated. This media and media/buffer after this step that are in contact with the 3H-cholesterol-labeled cells will be collected and disposed as 3H-radioactive liquid waste. Wash cells once with PBS. Incubate cells with DMEM + 0.2% fatty acid-free BSA overnight. Wash cells once with PBS. Incubate 0.5 ml of test samples (mouse HDL in DMEM + 0.2% BSA) with 3H-labeled cells for 4hrs @37ºC. Collect 150 µl supernatants and place into plastic scintillation vials containing 5 mL BioSafe II cocktail. Wash cells once with 1x PBS and lyse with 0.1N NaOH (500µl/well) @ room temperature. Collect 150 µl of the cell lysate and put into plastic scintillation vial containing 5 ml BioSafe II cockatail. Measure radioactivity by standard liquid scintillation counting. Calculation: Efflux (%) = 3H cpm of media * 100/ (3H cpm of media + 3H cpm of cell) The liquid 3H-radioactive waste will be separated from the dry 3H-radioactive waste and both will be disposed properly.

LCAT Assay: The LCAT activity assay kit was purchased from Roar Biomedical, Inc., New York, NY.
1. Prepare LCAT assay buffer: 150mM NaCl / 10 mM tris / 1 mM EDTA / 4 mM 2-mercaptoethanol. Adjust to pH 7.4
2. Reconstitute READ reagent with 99 ml 150 mM NaCl /10 mM tris / 1mM EDTA, pH 7.4.
3. Aliquot 200ml of the LCAT assay buffer into a clear walled 96-well plate.
4. Add 1ml plasma sample to the wells (use 10ml pipetmen to aliquot), or to blank, add 1ml PBS.
5. Add 1ml LCAT substrate reagent (in the dark) to each well, (not the blank), mix using the plate reader. Incubate at 37°C for 1 hour.
6. Aliquot 150ml of the READ reagent per well in a black walled 96-well plate. Add 50ml of incubated mixture per well. (mix using the plate reader mixing function for 15 seconds)
7. Read the fluorescent label at 340 nm excitation and emission at 390 nm (hydrolyzed) and 470 nm (unhydrolyzed).
8. A B6 plasma pool reference sample was included on each plate for normalization of the LCAT activity value.

Paraoxonase Assay: Substrate: Paraoxon, an oily liquid.
Caution: Extremely toxic, wear gloves, Hydrolyze waste in 10 M NaOH solution.
Assay Buffer: 2.0 M NaCl
0.1M Tris.Hcl, pH 8.5
2.0 mM CaCl2

2X Substrate solution: A 2.4 mM solution of paraoxon (Sigma, Cat # D-9286, Diethyl p-Nitrophenol Phosphate, 1g)  is made into the assay buffer. Use a screw capped plastic Falcon tube with a Poison sticker so it can be disposed of after use. 2.4 mM = 25 ul of paraoxon in 48.1 ml of assay buffer (enough for 2 x 96 well plates). Mix very well for at least 5 min (shaking by hand).

1. Aliquot 5 ml of plasma collected with heparin (20 units of heparin per 1 ml of blood) (NOT EDTA) into a well of a microtiter plate (Costar Cat. #: 9017, EIA/RIA 96-well flat bottom plate) that contains 95 ml of assay buffer. For each sample duplicate determinations are performed.

2. Set the plate reader (Molecular Devices, Model: Spectra Max 190) as

Function: Kinetics
Wavelength: 405 nm
Interval time: 15 sec
Total Time: 4 min

3. Using a multichannel pipetman, add 100 ml of 2X substrate solution into each well, put the plate into plate reader and start reading right away. The machine will compute the rate (mO.D./min) for you. Multiply the result with 200 to get mOD/min/ml of plasma.

4. To detoxify the unused portion of substrate solution, add 1/4 volume of 1M NaOH to the substrate solution and incubate at room temperature for 30 min before disposal.