Experimental Procedures

All experiments involving animals were conducted according to the UK Home Office regulations under valid personal and project licenses and in accordance with the Animal [Scientific Procedures] Act 1986. Experimental procedures were carried out in designated procedure rooms.

2.3.1 Identification of Experimental Cohorts

2.3.1.1 Ear Biopsy for identification and genotyping

For identification purposes ear biopsies were taken at weaning using a 2 mm ear punch (Harvard apparatus). DNA extraction from these biopsies was then used for genotyping. Biopsies were also taken from experimental cohorts at time of death to confirm genotype.

2.3.1.3 DNA extraction

Ear biopsies were collected and stored at -20°C until processing. Samples were then digested in 250 μL cell lysis buffer (VWR) containing 0.4 mg/mL proteinase K (Sigma) overnight at 42°C with agitation. The following day, samples were cooled to room temperature and 100 μL of protein precipitation solution (VWR) was added. The samples were then briefly mixed by inversion and insoluble debris and protein were pelleted by centrifugation at 16,000 G for 10 minutes at room temperature. The DNA was precipitated by adding the supernatant to 250 μL of isopropanol (ThermoFisher Scientific) in a DNase free eppendorf. The solution was mixed by inversion and DNA was pelleted by centrifugation at 16,000 G for 15 minutes. The supernatant was discarded and the pellet was washed with 70% ethanol. The DNA was pelleted by centrifugation at 16,000 G for 10 minutes and supernatant discarded. The pellet was air dried for 30 minutes at room temperature and resuspended in 250 μL of DNase/Rnase free water (Ambion). DNA was then stored at 4°C or at -20°C for long-term storage.

2.3.1.4 PCR Protocol

PCR was carried out in tube strips (0.2 mL thin walled, Alpha Laboratories). To each tube 3 μL of gDNA extracted from ear biopsies 47 µL of PCR mix (Table 2.2) was added. Primer sequences can be found in Table 2.3. The reaction was then run in a GS4 thermocycler (G storm) with the cycling times found in Table 2.2.

2.3.1.5 Gel electrophoresis of PCR products

Agarose gels were made by dissolving agarose (Bioline) 2% (w/v) in Tris- acetate-EDTA (TAE) buffer (National Diagnostic) and heating in a microwave at full power for 3 minutes and 10 seconds with agitation at regular intervals. After cooling slightly, 5 μL Safeview (NBS biologicals) was added per 100 mL. This was mixed gently to avoid bubbles and the gel was then cast into moulds with combs. Once set, the combs were removed and the gel placed into an electrophoresis tank and covered with 1x TAE. Following this 10 μL of each PCR sample was added to individual wells and run alongside a 100 bp ladder (PCR Biosystems). The samples were run at 120 V for approximately 30 minutes and then visualised using a GelDoc UV Transilluminator (Bio-Rad) and images taken with the GelDoc software (Bio-Rad). Genotypes were then established based on PCR product sizes in Table 2.3.

PCR Mix Pdx1-Cre KrasG12D EphA2tm1Jrui Rosa26tdRFP

Template DNA 3 µL 3 µL 3 µL 3 µL

Distilled Water 21.8 µL 21.7 µL 21.7 µL 21.7 µL

2x Hot Start Taq Mix

Red (PCR Biosystems) 25 µL 25 µL 25 µL 25 µL Primer 1 (100 mM) 0.1 µL 0.1 µL 0.1 µL 0.1 µL Primer 2 (100 mM) 0.1 µL 0.1 µL 0.1 µL 0.1 µL Primer 3 (100 mM) N/A 0.1 µL 0.1 µL 0.1 µL PCR Cycling conditions

Initial Denaturation 3 min; 94°C 3 min; 94°C 3 min; 94°C 3 min; 94°C

Cycle number 35 35 35 35

Step 1: Denaturation

(Time; Temp) 30 sec;94 °C 30 sec;94 °C 30 sec;94 °C

30 sec;94 °C

Step 2: Annealing

(Time; Temp) 30 sec; 55°C 30 sec;68 °C 30 sec; 60°C

30 sec; 56°C

Step 3: Extension

(Time; Temp) 1min; 72°C 1min; 72°C 1min; 72°C 1min; 72°C

Final 5min; 72°C 5min; 72°C 5min; 72°C 5min; 72°C

Hold at 4°C Hold at 4°C Hold at 4°C Hold at 4°C

Gene Forward primer (5' - 3') Reverse primer (5' - 3') Product Size

Pdx1-Cre CTG GAC TAC ATC TTG AGT

TGC

GGT GTA CGG TCA

GTA AAT TTG 650 bp

KrasG12D

WT Forward: TGT CTT TCC

CCA GCA CAG T CTG CAT AGT ACG CTA

TAC CCT GT

WT = 250bp Mut Forward: GCA GGT CGA

GGG ACC TAA TA Mutant=100bp

EphA2tm1Jrui TGT CAC TTG CGA ACA _{GTG CT}

WT: CGC TAT CAC ACT

CAG CAG GA WT=414 bp

Mutant: GTG GAG AGG

CTT TTT GCT TC Mutant=280bp Rosa26tdRFP AAG ACC GCG AAG AGT

TTG TCC

Mutant: GTG GAG AGG

CTT TTT GCT TC WT = 209 bp WT: TAA GCC TGC CCA

GAA GAC TCC Mutant = 310 bp

Table 2.3 Primer sequences used for genotyping and product sizes. KrasG12D, EphA2 and RFP genotyping also includes primer for wild-type (WT) and mutant alleles.

2.4 Experimental Cohorts induction

After PCR was used to identify the genotype of mice they were randomly placed into control and experimental cohorts at 42 – 56 days of age. Cre- recombinase translocation to the nucleus and hence recombination was induced by intraperitoneal injection (IP) of tamoxifen. Stocks of 10 mg/mL tamoxifen (Sigma) were generated by dissolving tamoxifen in corn oil (Sigma) overnight at 37°C with agitation. This was then diluted 1:1000 to produce 10 μg/mL of tamoxifen. Animals were then administered 100 μL of 10 μg/mL in a single injection to produce a 1 μg dose respectively. For high dose tamoxifen was dissolved in corn oil at 20 mg/mL and dissolved in the same manner as before. Mice were then injected with 9 mg/40 g of body weight every other day for 3 injections over 5 days.