Gene Expression Validation in apoE-/- Mice Protocol
Phase 1: apoE-/- Mouse Breeding and Experimental Groups (Days 1-28)
Mouse Maintenance and Diet: Obtain apoE-/- mice (C57BL/6J-Apoe<tm1Unc>/J, JAX #002052) and C57BL/6J wild-type controls. House on either: (a) normal chow (NC, 4% fat, Protocol #5VO5), or (b) Western diet (WD, 21% milk fat, 0.2% cholesterol, Harlan Teklad #TD.88137) for 8 or 16 weeks. Randomize at weaning (3 weeks).
Experimental Groups: (a) WT + NC (n=10), (b) WT + WD (n=10), (c) apoE-/- + NC (n=12), (d) apoE-/- + WD (n=15). Match groups by sex distribution. Record body weight and food intake weekly.
...APOE--mice-protocol" style="color:#4fc3f7;margin:1.5rem 0 0.6rem;font-size:1.15rem;font-weight:700;border-bottom:2px solid rgba(79,195,247,0.3);padding-bottom:0.3rem">Gene Expression Validation in apoE-/- Mice Protocol
Phase 1: apoE-/- Mouse Breeding and Experimental Groups (Days 1-28)
Mouse Maintenance and Diet: Obtain apoE-/- mice (C57BL/6J-Apoe<tm1Unc>/J, JAX #002052) and C57BL/6J wild-type controls. House on either: (a) normal chow (NC, 4% fat, Protocol #5VO5), or (b) Western diet (WD, 21% milk fat, 0.2% cholesterol, Harlan Teklad #TD.88137) for 8 or 16 weeks. Randomize at weaning (3 weeks).
Experimental Groups: (a) WT + NC (n=10), (b) WT + WD (n=10), (c) apoE-/- + NC (n=12), (d) apoE-/- + WD (n=15). Match groups by sex distribution. Record body weight and food intake weekly.
Tissue Collection: Fast mice 4 hours, collect plasma (EDTA, via cardiac puncture under isoflurane anesthesia). Perfuse with PBS. Dissect liver, aorta (arch to iliac bifurcation), spleen, and mesenteric adipose. Snap-freeze in liquid nitrogen or fix in 4% PFA.
Phase 2: RNA Extraction and Gene Expression Profiling (Days 29-42)
RNA Extraction from Liver: Homogenize 50 mg liver in Trizol (1 mL, 30 sec, bead disruptor). Extract total RNA per manufacturer protocol. Assess quality via Bioanalyzer (RIN ≥ 8.0 for all samples). Treat with DNase I (DNA-free kit). Verify purity (A260/280 ≥ 1.9, A260/230 ≥ 1.8).
qRT-PCR Array for Complement/Inflammation Genes: Run TaqMan Array Mouse Cardioid 96-well plates (Applied Biosystems #4413277) covering 84 genes: complement system (C1QA, C1QB, C1QC, C3, C5), inflammatory cytokines (IL1B, IL6, TNF, CCL2), endothelial function (CDH5, KLF2, NOS3), lipid metabolism (LDLR, APOE, ABCA1). Include housekeepers (GAPDH, HPRT1).
Individual Gene Validation: Validate top differentially expressed targets via individual TaqMan assays (C1QA #Mm00432142_m1, C1QC #Mm01150960_g1, SPI1 #Mm00453882_m1). Run on QuantStudio 7 Flex. Calculate fold change via ΔΔCt method (2^-ΔΔCt). Normalize to HPRT1.
Phase 3: Protein and Histological Validation (Days 43-56)
Complement Protein Measurement: Measure C1QA and C1QC protein in liver homogenates via ELISA (C1QA: Abcam #ab200007, C1QC: Cusabio #CSB-EL004562MO). Normalize to total protein (BCA assay). Run in duplicate with standard curve.
Immunofluorescence Staining: Fix frozen liver sections (8 μm) in acetone (-20°C, 10 min). Block with 5% normal donkey serum. Stain C1QA (1:100, Abcam #ab90442), SPI1 (PU.1, 1:100, Abcam #ab184935), and CD68 (macrophage marker, 1:200, Abcam #ab783). Detect with Alexa Fluor 488/594 secondaries. Image on Zeiss LSM 880 confocal (20×).
Morphometric Analysis: Quantify C1QA+ area fraction in liver sections using ImageJ. Correlate with gene expression (qRT-PCR) and protein levels (ELISA) via Pearson correlation.