H/M Genotyping Protocol: MMRRC Line 11858 Strain Characteristics: Human mutant hepatic lipase transgene (HL-S145G), hepatic lipase knockout, apolipoprotein E knockout. Maintained homozygous. Hepatic Lipase Transgene Assay Type: Allelic quantification PCR assay can detect homozygous from heterozygous animals. Heterozygous animals not available for testing at time of assay development. DNA Extraction: DNA Extraction: DNA from tail snips was extracted using Qiagen’s DNeasy kit. Kit directions for animal tissues were performed with a few minor modifications as follows: RNase treatment for 20 minutes was performed after overnight digestion of tissue sample, DNA was eluted in 200 μl of AE buffer once. Use controls that have also been RNase treated. Primer Information: Genomic position: This assay generates a 78 bp product from the NM_000236.1 transcript. Actual primer information is undisclosed from Applied Biosystems. Real-time PCR Master Mix Components: component ABI's Taqman PCR master Mix Hs00165106 20X Assay Mix nuclease free water manufacturer Applied Biosystems Applied Biosystems concentration 2X 20X μl/rxn 25 2.5 12.5 PCR Setup: Final Reaction: 45μl master mix & 5μl DNA template (20ng/μl DNA concentration). Check concentration of diluted DNA before setting up assay. All reactions were performed in 200μl thin walled optical grade PCR tubes and were run in Applied Biosystems 7000 Real-time PCR system. Cycle Parameters: o 1) 50 C 2 minutes o 2) 95 C 10 minutes o 3) 95 C 15 sec o 4) 60 C 1 minute 5) Repeat steps 3-4 39 times for a total of 40 cycles Product Analysis: Ct-22.63-24.37 cycles for transgene positive mice compared to WT mice with no amplification (no cycles). Heterozygous animals were not available for testing. Hepatic Lipase Knockout Assay Type: PCR - can distinguish heterozygous animals from homozygous animals. Heterozygous animals not available for testing at time of assay development. DNA Extraction: DNA Extraction: DNA from tail snips was extracted using Qiagen’s DNeasy kit. Kit directions for animal tissues were performed with a few minor modifications as follows: RNase treatment for 20 minutes was performed after overnight digestion of tissue sample, DNA was eluted in 200 μl of AE buffer once. Use controls that have also been RNase treated. Primer Information: 1) Name: HL-F 2) Name: HL-R Sequence: 5'-AAT CTG CGA AGT TTT CTC GG-3’ Sequence: 5'-TTC TTC CAA TCT TGT TCT TCC C-3’ Genomic location: Chromosome 9: 70850973:70851592:-1 PCR Master Mix Components: component manufacturer concentration μl/rxn buffer Roche 10X 2 dNTP Roche 1.25mM 3.2 HL-F IDT 25μM 0.3 HL-R IDT 25μM 0.3 FastStart taq Roche 5 U/μl 0.2 nuclease free water PCR Setup: Final Reaction: 13 19μl master mix & 1μl DNA template All reactions were performed in 200μl thin walled PCR tubes and were run in Perkin Elmer 2400 thermocycler or Applied Biosystems 2700 thermocycler. Cycle Parameters: 1) 2) 3) 4) 5) 6) 7) 94oC 5 minutes 94oC 30 seconds 59oC 30 seconds 72oC 2 minutes Repeat steps 2-4 34 times for a total of 35 cycles 72oC 7minutes 4oC hold until refrigerate product Product Analysis: All products were analyzed on a 1% agarose gel with ethidium bromide staining Expected products: WT: 111 bp Hom: 1200-1300 bp Het: 111 bp and 1200-1300 bp Apolipoprotein E Knockout Assay Type: PCR – can distinguish heterozygous animals from homozygous animals. Heterozygous animals not available for testing at time of assay development. DNA Extraction: DNA Extraction: DNA from tail snips was extracted using Qiagen’s DNeasy kit. Kit directions for animal tissues were performed with a few minor modifications as follows: RNase treatment for 20 minutes was performed after overnight digestion of tissue sample, DNA was eluted in 200 μl of AE buffer once. Use controls that have also been RNase treated. Primer information: based on The Jackson Lab’s protocol for strain 002052 (Apoetm1Unc) at http://jaxmice.jax.org/pub-cgi/protocols/protocols.sh?objtype=protocol&protocol_id=221. 1) Name: oIMR180 2) Name: oIMR181 3) Name: oIMR182 Sequence: 5'- GCC TAG CCG AGG GAG AGC CG-3’ Sequence: 5'- TGT GAC TTG GGA GCT CTG CAG C-3’ Sequence: 5'- GCC GCC CCG ACT GCA TCT-3’ Primer location: oIMR180: Forward (5'-3') primer corresponding to Apoe exon 3 genomic sequence (nucleotides 2406-2425 in Genbank #D00466. oIMR181: Reverse (3'-5') primer from Apoe exon 3 (corresponds to nucleotides 25602539 of Genbank # D00466). oIMR182: Reverse primer. Primer corresponds to region within the vector used to disrupt the Apoe gene. PCR Master Mix Components: component manufacturer concentration μl/rxn buffer Roche 10X 2 dNTP Roche 1.25mM 3.2 oIMR180 IDT 25μM 0.3 oIMR181 IDT 25μM 0.3 oIMR182 IDT 25μM 0.3 FastStart taq Roche 5 U/μl 0.2 nuclease free water PCR Setup: Final Reaction: 12.7 19μl master mix & 1μl DNA template All reactions were performed in 200μl thin walled PCR tubes and were run in Perkin Elmer 2400 thermocycler or Applied Biosystems 2700 thermocycler. Cycle Parameters: 1) 2) 3) 4) 5) 6) 7) 5 minutes 94oC 94oC 30 seconds 66oC 40 seconds 72oC 2 minutes Repeat steps 2-4 34 times for a total of 35 cycles 72oC 7minutes 4oC hold until refrigerate product Product Analysis: All products were analyzed on a 3% agarose gel with ethidium bromide staining Expected products: WT = 155 bp Hom = 245 bp Het = 155 and 245 bp