Mouse colonies

2.2.1 Mouse husbandry

All mouse strains were maintained at the Australian Phenomics Facility at The Australian National University according to Australian Standards for Animal Care under ethics protocol A2015/10. Mice were maintained in a light cycle of 12 h light: 12 h dark, the midpoint of the dark cycle being 12 a.m. For the production of staged embryos, 12 p.m. on the day of the appearance of the vaginal plug is designated 0.5 dpc.

2.2.2 Low-quality genomic DNA preparation

Low-quality genomic DNA preparation was routinely used for genotyping of either ear biopsy tissue or embryonic tissue. Ear notches were collected in 50 μL of lysis solution [50 mM Tris(hydroxymethyl)aminomethane (Tris)-HCl (pH 8.5), 1 mM Ethylenediaminetetraacetic acid (EDTA), and 5% Tween 20] containing 2 μL of Proteinase K (10 μg/μL; Sigma Aldrich, Cat. No. P2308) and incubated at 55°C for 60 minutes, followed by 95°C for 10 minutes to inactivate Proteinase K. Tissue debris was pelleted by centrifuging at 2,000 g for 5 minutes and each sample was diluted 1:10 in Analar H2O. Embryos were genotyped using a fragment of extra embryonic

tissue/ectoplacental cone (7.5 dpc) or yolk sac (8.5 dpc and 9.5 dpc). Tissue was collected in 20 μL (7.5 dpc), 25 μL (8.5 dpc) or 35 μL (9.5 dpc) of lysis buffer (50 mM Tris- HCl, pH 8.5, 1 mM EDTA, 0.5% Tween 20) containing 1 μL of Proteinase K (10 μg/μL), followed by incubation at 55°C for 25 minutes and 95°C for 5 minutes to inactivate Proteinase K.

2.2.3 High-quality genomic DNA preparation

Approximately 0.5 cm of the tail or one ear were biopsied and homogenised in 1.5 mL of TE-SDS (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1% SDS) containing 0.5 µg/µL proteinase

49 K (PK) in a 12 mL glass corex tube on ice. The sample was sealed and incubated overnight at 55o_{C. Cellular debris was precipitated by the addition 8M KOAc to a final}

concentration of 1.2 M, and the specimen was centrifuged in a Sorvall HB-4 rotor at (23,000 g, 4o_{C) for 20 minutes to pellet debris. To precipitate the DNA 1.5 mL of the}

supernatant transferred to 3.75 mL of 100% EtOH in a 15 mL Falcon tube. The sample was inverted 10 times, and the DNA precipitate removed by spooling onto a glass Pasteur pipette with a flame sealed end. DNA was washed by immersion in 1 mL of 70% EtOH, allowed to air dry, after which it was resuspended in 500 µL of TE. DNA was quantified by measurement of the optical density at 260 nm via NanoDrop (NanoDrop 1000 v8.1) DNA preparations were diluted to 10 ng/µL for further PCR analysis.

2.2.4 Genotyping assays

Genotyping of adults and embryos from the ENU, katun, batface and atp1a1 mouse strains was done using the following techniques:

2.2.4.1High resolution melt analysis (HRMA)

Digested ear biopsies were genotyped using a previously described High Resolution Melt Analysis (HRMA) assay (Thomsen et al., 2012) with no deviations. HRMA genotyping primers are shown in Table 2.2.

2.2.4.2Allelic discrimination

PCR for allelic discrimination was done using TaqMan® Universal PCR Master Mix (Life Technologies; Cat. No. 4304437). All reactions were carried out in a final volume of 15 μL with ~30 ng of digested ear notch DNA in the presence of 0.9 μM of primers (Table 2.2). Additionally 0.25 μM of allele-specific probes (Applied Biosystems®) were used to identify the wild-type alleles and mutant allele (Figure 2.1, Table2.4). Reactions were set up in 96-well Half-Skirted PCR Microplates (Axygen®; Cat. No. PCR-96-LP-AB-C) covered

50 with Axygen Microplate Sealing Film (Fisher Scientific; Cat. No. UC500) and performed using the StepOnePlus™ Real-Time PCR System (Applied Biosystems®). The StepOne Software (version 2.2.2; Applied Biosystems®) was used to run the assay using the following conditions: an initial pre-PCR read at 60°C for 30 seconds to record background fluorescence, followed by 95°C for 10 minutes to denature the template and a cycling stage of 95°C for 15 seconds and of 60°C for 1 minute for 50 cycles. A post-PCR read was performed at 60°C for 30 seconds to collect data after completion of the PCR. Data was analysed using the same software that records the pre- and post-PCR reads and calculates normalised dye fluorescence (∆Rn) from the wild-type and mutant alleles as a function of cycle number. Based on this data the software called the sample as homozygous for either wild-type or mutant allele, or heterozygous with both alleles.

51 Figure 2.1: Allelic discrimination using fluorogenic probes. Wild-type and mutant alleles were distinguished using non-extendable oligonucleotide fluorogenic probes (Livak, 1999). The FAM™ (6-carboxyfluorescein) probe complemented the wild-type allele, while the VIC® (4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein) probe complemented the mutant allele. The probes differed by only one nucleotide (at the site of the mutation lucs), and at the 5' end were labelled with the reporter (VIC® or FAM™) dye and at the 3' end with the quencher (MGBNFQ: minor groove binding non-fluorescent quencher). The proximity of the reporter dye to the quencher reduced fluorescence via the reporter. During PCR as the forward primer extended the target specific probe was degraded by the 5'-3' exonuclease activity of the Taq DNA polymerase (Arya et al., 2005), which released the reporter dye allowing it to fluoresce. Both probes were included in the same well for each PCR reaction thus fluorescence was only observed when target allele for the probe was present (McGuigan et al., 2002). As PCR cycles increased, the fluorescence intensity increased that was proportional to the accumulation of the PCR product. The fluorescence was measured using a Real-Time PCR machine. This figure was adapted from Applied Biosystems’ Allelic Discrimination Getting Started Guide.

52 Table 2.4: Probes used for allelic discrimination.

wt: wildt-ype, mt: mutant, FAM™: 6-carboxyfluorescein, VIC: 4,7,2'- trichloro-7'-phenyl-6-carboxyfluorescein and MGBNFQ: minor groove binding non-fluorescent quencher.

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