TITLE: Multiplex Genotyping PCR for the KPC Pancreatic Cancer Mouse Model
AUTHORS: Shou Kitahara, Shounak Ranabhor, Thae Su Thu, Neha Ramesh, Chang-il Hwang

[DESCRIPTION]
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.

[STEPS]
SECTION: Crude genomic DNA isolation
1. 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)
SECTION: Crude genomic DNA isolation
2. Add 3 µl Proteinase K (20 mg/mL) to 1 mL of Lysis buffer prior to use (Store in -20°C)
SECTION: Crude genomic DNA isolation
3. Add 30 µl Lysis buffer with Proteinase K to cut tissue in 1.5 mL tube
SECTION: Crude genomic DNA isolation
4. Incubate at 56°C for 1.5 hours
SECTION: Crude genomic DNA isolation
5. Place on ice briefly and spin down briefly in a tabletop centrifuge 
SECTION: Crude genomic DNA isolation
6. Incubate at 96°C for 10 min in ThermoMixer
SECTION: Polymerase Chain Reaction for KPC
7. 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 
SECTION: Polymerase Chain Reaction for KPC
8. Aliquot 19 µL of master mix into each PCR tube
SECTION: Polymerase Chain Reaction for KPC
9. Add 1 µL DNA template to individual PCR tubes
SECTION: Polymerase Chain Reaction for KPC
10. 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 ∞ 
SECTION: Polymerase Chain Reaction for Trp53 Het/Homo
11. 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
SECTION: Polymerase Chain Reaction for Trp53 Het/Homo
12. Aliquot 19 µL of master mix into each PCR tube
SECTION: Polymerase Chain Reaction for Trp53 Het/Homo
13. Add 1 µL DNA template to individual PCR tubes
SECTION: Polymerase Chain Reaction for Trp53 Het/Homo
14. 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 ∞ 
SECTION: Gel Electrophoresis
15. Make 2% agarose gel in TAE buffer
SECTION: Gel Electrophoresis
16. Add 2 µL ethidium bromide (10 mg/ml stock concentration) to 100 ml melted agarose. Swirl to mix.
SECTION: Gel Electrophoresis
17. Pour into the casting mold with combs inserted to set up wells.
SECTION: Gel Electrophoresis
18. Remove combs and casting mold from agarose gel after the cast harden (~ 30 min)
SECTION: Gel Electrophoresis
19. Place the gel into electrophoresis apparatus 
SECTION: Gel Electrophoresis
20. Add 1X TAE buffer to electrophoresis apparatus until the gel is submerged
SECTION: Gel Electrophoresis
21. Add 4 µL 6X loading dye to each 20 µL PCR product and load 10 µL of it into the wells
SECTION: Gel Electrophoresis
22. Load 3 µL of 100 bp DNA ladder mixed with loading dye to 1st well of each lane
SECTION: Gel Electrophoresis
23. Run gel at 120V until loading dye is visualized 75-80 % down the gel
SECTION: Gel Electrophoresis
24. Remove the gel from the electrophoresis apparatus and visualize it under UV light
SECTION: Gel Electrophoresis
25. Analyze images to determine the genotype for each mouse