Mar 30, 2023
Multiplex Genotyping PCR for the KPC Pancreatic Cancer Mouse Model
- Shou Kitahara1,
- Shounak Ranabhor1,
- Thae Su Thu1,
- Neha Ramesh1,
- Chang-il Hwang1
- 1University of California Davis
- Shou Kitahara: SK and SR contributed equally to this protocol.
- Chang-il Hwang: Corresponding author
External link: https://cihwang.com
Protocol Citation: Shou Kitahara, Shounak Ranabhor, Thae Su Thu, Neha Ramesh, Chang-il Hwang 2023. Multiplex Genotyping PCR for the KPC Pancreatic Cancer Mouse Model. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly7bxrlx9/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 30, 2023
Last Modified: March 30, 2023
Protocol Integer ID: 79723
Keywords: pancreatic cancer, PDAC, KPC, genotyping, multiplex, Kras, Trp53, Pdx1-Cre
Funders Acknowledgement:
National Cancer Institute
Grant ID: R37CA249007
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Abstract
Genetically engineered mouse model (GEMM) is one of the most important pre-clinical models in cancer research. In pancreatic cancer research, Kras+/LSL-G12D; Trp53+/LSL-R172H; Pdx-1-Cre (KPC) mouse model has been widely used in the laboratory setting, since it faithfully recapitulates the progression of human pancreatic ductal adenocarcinoma. Polymerase Chain Reaction (PCR) is commonly used to genotype GEMMs. However, genotyping one gene at a time is inefficient and labor intensive. To simplify the genotyping process, we multiplexed three separate genotyping PCR protocols for a single PCR reaction. In addition, we provided the optimized PCR protocol for determining heterozygous or homozygous status of Trp53LSL-R172Hallele. Overall, this protocol offers cost-efficient and accurate genotyping results for KPC mice.
Guidelines
Introduction
Genetically engineered mouse models (GEMMs) are critical preclinical models that allow researchers to investigate disease progression and therapeutic responses in vivo. In cancer specifically, GEMMs allow researchers to study the means of tumor progression in patients, including tumor initiation, metastasis, and mutational heterogeneity, by aptly recapitulating human cancer characteristics1. To use GEMMs in the laboratory setting, it is necessary to cross individual genetically modified alleles and maintain the desired genotypes.
Mutations in KRAS (Kirsten rat sarcoma viral oncogene homolog) and TP53 are the two most frequently mutated genes in cancers2.For pancreatic ductal adenocarcinoma (PDAC), KRAS mutations are present in about 95% of cases and TP53 are co-present in about 70% of cases3. Therefore, mice with Kras+/LSL-G12D; Trp53+/LSL-R172H; Pdx1-Cre (KPC) are widely used because this model faithfully recapitulates human pancreatic cancer pathogenesis4.
It is common practice to isolate genomic DNA from toes, tails, or ears and perform molecular methods like PCR (polymerase chain reaction), Southern blot or dot blot techniques5. In general, the genotyping methods should be rapid and reproducible, and allow for the analysis of large numbers of mice. However, it is still labor-intensive to genotype multiple alleles individually. Mis-genotyping of the alleles could lead to irrevocable consequences6.
Here, we have provided a simple, rapid, and cost-effective multiplex genotyping protocol for KPC mice with crude DNA extracts from tail, toes, or ear biopsy. In addition, we refined the protocol to distinguish the homozygous (homo) alleles for Trp53LSL-R172H from heterozygous (het). This protocol requires a single PCR reaction for three alleles of K (KrasLSL-G12D), P (Trp53LSL-R172H) and C (Cre) as well as an internal positive control without any commercial genomic DNA isolation kit.
Materials
- Crude genomic DNA isolation
●Lysis buffer
i.10 mM Tris-HCL
ii.50 mM KCl
iii.2.5 mM MgCl2
iv.0.45 % NP-40 Buffer
v.0.45 % Tween 20
vi.60 µg/mL Proteinase K (20 mg/mL Proteinase K, NEB, #P8107)
●ThermoMixer (Eppendorf, Cat #5382000023) or water bath
2. Polymerase Chain Reaction for KPC Genotyping
●Platinum hot start PCR 2X mix (Thermo Fisher Scientific, Cat # 13000012),
●Template DNA (retrieved from toe/tail DNA isolation)
●7 primer sequences as detailed in Table 1 and 2
●UltraPure™ DNase/RNase-Free Distilled Water(Thermo Fisher Scientific (Invitrogen), Cat # 10977015)
3. Polymerase Chain Reaction for Trp53 Het/Homo Genotyping
●MiniAmp Thermal Cycler (Thermo Fisher Scientific)
●Template DNA (retrieved from toe/tail DNA isolation)
●10 mM Deoxynucleotide (dNTP) Solution Mix (NEB, #N0447S)
●10 mM primer (4 primers)
●NEB Taq polymerase (NEB, #M0273) and Standard buffer
●UltraPure™ DNase/RNase-Free Distilled Water(Thermo Fisher Scientific (Invitrogen), Cat # 10977015)
4. Gel Electrophoresis
●100 bp ladder (NEB, N3231)
●Ethidium bromide (10 mg/mL, Sigma, E1510)
●Agarose
●1X TAE Buffer
i.242g Tris free base (Thermo Fisher Scientific, Cat # BP152-5)
ii.18.61g Disodium EDTA (Thermo Fisher Scientific, Cat # BP120-1)
iii.57.1 ml Glacial Acetic Acid (Thermo Fisher Scientific, Cat #A38-212)
iv.Double-distilled H2O to 1L
v.Dilute 50X to desired volume of 1X with Double-distilled H2O
●Gel casting mold and combs
●Electrophoresis apparatus
●6X Loading Dye (NEB, #B7025)
●Power Supply
Table 1. KPC Primers
| Primer Type | Sequence 5’ → 3’ | |
| Internal Positive Control Forward | CTG TCC CTG TAT GCC TCT GGT CGT A | |
| Internal Positive Control Reverse | AGA TGG AGA AAG GAC TAG GCT ACA ACT TAC | |
| Cre Forward | GGA TCG CCA GGC GTT TTC TG | |
| Cre Reverse | CCA GCC ACC AGC TTG CAT GA | |
| LSL-Forward | AGC TAG CCA CCA TGG CTT GAG TAA GTC TGC A | |
| Kras Reverse | CCT TTA CAA GCG CAC GCA GAC TGT AGA | |
| Trp53 Reverse | CTT GGA GAC ATA GCC ACA CTG |
Table 2. Trp53 Het/Homo Primers
| Primer Type | Sequence 5’ → 3’ | |
| Internal Positive Control Forward | CAA ATG TTG CTT GTC TGG TG | |
| Internal Positive Control Reverse | GTC AGT CGA GTG CAC AGT TT | |
| Trp53 1loxp Forward | AGC CTG CCT AGC TTC CTC AGG | |
| Trp53 1loxp Reverse | CTT GGA GAC ATA GCC ACA CTG |
Figure 1. Representative gel image of KPCgenotyping results. PCR was used to amplify KrasLSL-G12D ~ 550 bp. Trp53LSL-R172H ~ 270 bp, Pdx1-Cre~ 800 bp, Internal positive control ~ 415 bp, and negative control - no band.
Figure 2. Representative gel image of genotyping of heterozygous vs. homozygous Trp53LSL-R172Halleleprotocol. PCR was used to amplify Heterozygous Trp53+/LSL-R172H~ 270 bp, Homozygous Trp53LSL-R172H/LSL-R172H~ no band, Internal positive control ~ 200 bp and negative control - no band.
Crude genomic DNA isolation
Crude genomic DNA isolation
Prepare Lysis Buffer (Recipe for 10 mL)
●0.0121 g Tris-HCl pH 8.0
●0.0373 g KCl
●12.5 µl 2M MgCl2
●45 µL NP-40
●45 µL Tween-20
●9.9 mL Ultrapure Distilled Water (DW)
Add 3 µl Proteinase K (20 mg/mL) to 1 mL of Lysis buffer prior to use (Store in -20°C)
Add 30 µl Lysis buffer with Proteinase K to cut tissue in 1.5 mL tube
Incubate at 56°C for 1.5 hours
Place on ice briefly and spin down briefly in a tabletop centrifuge
Incubate at 96°C for 10 min in ThermoMixer
Polymerase Chain Reaction for KPC
Polymerase Chain Reaction for KPC
Prepare PCR master mix for KPC in 1.5mL centrifuge tubes
- 10 µL Platinum hot start PCR Mix
- 0.4 µL 10 mM primer each (7 primers)
- 6.2 µL DW
Aliquot 19 µL of master mix into each PCR tube
Add 1 µL DNA template to individual PCR tubes
- Perform PCR using the following conditions:
a.94°C 3 min
b.94°C 1 min/ 65°C 2 min/ 72°C 1 min (40 cycles)
c.72°C 3 min / 4°C ∞
Polymerase Chain Reaction for Trp53 Het/Homo
Polymerase Chain Reaction for Trp53 Het/Homo
- Prepare PCR master mix for Trp53 het/homo in 1.5mL centrifuge tubes
- 2 µL 10X NEB polymerase buffer
- 0.4 µL 10mM dNTP
- 0.4 µL 10mM primer (4 primers)
- 0.1 µL Taq polymerase
- 14.9 µL DW
Aliquot 19 µL of master mix into each PCR tube
Add 1 µL DNA template to individual PCR tubes
- Perform PCR using the following conditions:
a.94°C 3 min
b.94°C 30sec/ 60°C 30sec/ 72°C 30sec (40 cycles)
c.72°C 3 min / 4°C ∞
Gel Electrophoresis
Gel Electrophoresis
Make 2% agarose gel in TAE buffer
Add 2 µL ethidium bromide (10 mg/ml stock concentration) to 100 ml melted agarose. Swirl to mix.
Pour into the casting mold with combs inserted to set up wells.
Remove combs and casting mold from agarose gel after the cast harden (~ 30 min)
Place the gel into electrophoresis apparatus
Add 1X TAE buffer to electrophoresis apparatus until the gel is submerged
Add 4 µL 6X loading dye to each 20 µL PCR product and load 10 µL of it into the wells
Load 3 µL of 100 bp DNA ladder mixed with loading dye to 1st well of each lane
Run gel at 120V until loading dye is visualized 75-80 % down the gel
Remove the gel from the electrophoresis apparatus and visualize it under UV light
Analyze images to determine the genotype for each mouse
Protocol references
References
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2 Mendiratta, G. et al. Cancer gene mutation frequencies for the U.S. population. Nat Commun12, 5961 (2021). https://doi.org:10.1038/s41467-021-26213-y
3 Kim, M. P. et al. Oncogenic KRAS Recruits an Expansive Transcriptional Network through Mutant p53 to Drive Pancreatic Cancer Metastasis. Cancer Discov11, 2094-2111 (2021). https://doi.org:10.1158/2159-8290.CD-20-1228
4 Hingorani, S. R. et al. Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice. Cancer Cell7, 469-483 (2005). https://doi.org:10.1016/j.ccr.2005.04.023
5 Cinelli, P., Rettich, A., Seifert, B., Burki, K. & Arras, M. Comparative analysis and physiological impact of different tissue biopsy methodologies used for the genotyping of laboratory mice. Lab Anim41, 174-184 (2007). https://doi.org:10.1258/002367707780378113
6 Lloyd, K., Franklin, C., Lutz, C. & Magnuson, T. Reproducibility: Use mouse biobanks or lose them. Nature522, 151-153 (2015). https://doi.org:10.1038/522151a
7 Donehower, L. A. et al. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature356, 215-221 (1992). https://doi.org:10.1038/356215a0