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