Discusión 58

2.9.1 Genomic DNA Lysis

Adult mice were genotyped using ear biopsy DNA. A ~20 mm2 _{notch of ear was taken from each}

mouse. To each ear notch sample, 50 µL of lysis solution (50 mM Tris-HCl pH 8.5, 1 mM EDTA, 5.0% Tween 20) and 2 µL of Proteinase K (PK; 10 mg/mL in dH2O) was added and incubated at

55 °C for 60 minutes, followed by 95 °C for 10 minutes to inactivate the PK. Tissue debris was pelleted by centrifuging at 2000 x g for 5 minutes, and each sample diluted 1:10 in AnalaR H2O.

Embryos were genotyped using a fragment of extra ectoplacental cone (7.5 dpc), amnion or yolk sac (³ 8.5 dpc). To each embryo sample, 10 μL of lysis solution and 2 μL of PK was added and samples incubated at 55 °C for 25 minutes and 95 °C for 5 minutes to inactivate the PK. Tissue debris was pelleted by centrifuging at 2000 x g for 5 minutes, and each sample diluted 1:4 in AnalaR H2O.

2.9.2 Genotyping PCRs

To genotype transient PiggyBac transgenic embryos, oligonucleotide RA1058 was paired with oligonucleotide RA1547 (pBB262-NCE-lacZ wildtype and mutant), RA1548 (pBB262-lacZ) and RA1059 (pBB262-mNet-lacZ). All reactions were carried out in a 10 μL volume with a final concentration of 0.6 μM of each oligonucleotide and 2X PCR ReddyMix™ Master Mix without KCl (Table 2.2). 1 M Betaine was included for pBB262-NCE-lacZ genotyping. PCRs to be analysed via gel electrophoresis were performed in 96-Well Clear, Flat Top PCR plates (Axygen; Cat. No. PCR-96-FLT-C). All PCRs were performed in an Eppendorf Mastercycler® using the TD70 Touchdown PCR programs (Table 2.12) and the products run on a 1.5% agarose gel. The production of a 443 bp (pBB262-mNet-lacZ), 561 bp (pBB262-NCE-lacZ wildtype and mutant) and 366 bp (pBB262-lacZ) were deemed a positive genotype.

Genotyping of CRISPR 3’UTR mutant embryos was performed by Nay Chi of the JCSMR Transgenesis Facility. Briefly, all reactions were carried out in a 50 μL volume with a final concentration of 0.6 μM of each oligonucleotide (RA1778 and RA1779, Appendix Table A1.2), 2X MyTaq™ HS Mix (Bioline; Table 2.2) and 1 M Betaine. PCRs to be analysed via gel electrophoresis were performed in 96-Well Clear, Flat Top PCR plates (Axygen; Cat. No. PCR-96-FLT-C). All PCRs were performed in an Eppendorf Mastercycler® using a hot start PCR program according to the manufacturer’s instructions. The products run on a 1% agarose gel; an amplicon of 1019 bp indicated a wildtype embryo.

2.9.3 High Resolution Melt Assay (HRMA)

Genomic DNA extracted from ear notches of adult mice and embryo tissue were genotyped using HRMA, as described by (Thomsen et al., 2012). PCRs were carried out using ImmoMix (Bioline; Cat. No. BIO-25020) and included the LC Green® Plus+ Melting Dye (Idaho Technology Inc.; Cat. No. BCHM-ASY-0005). All reactions were carried out in a final volume of 10 μL with ~30 ng of digested ear notch DNA, with a final concentration of 0.6 μM of each oligonucleotide (Appendix Table A1.2). Reactions were set up in Hard-Shell® 96-well PCR Plates (BioRad; Cat. No. HSP-9665) covered with Axygen Microplate Sealing Film (Fisher Scientific; Cat. No. UC500). To avoid evaporation during the HRMA process, each reaction was covered with ~10 μL of mineral oil prior to PCR. All PCRs were performed in an Eppendorf Mastercycler® using three Touchdown

PCR programs: TD60, TD65 and TD70 (Table 2.12). On completion of a PCR reaction, the 96-well plate containing PCR products was placed directly into a Light Scanner HR 96 (Idaho Technologies Inc.) and samples melted from 60 °C to 95 °C at a rate of 0.1 secs-1_{. The LC Green®}

dye specifically binds to double-stranded DNA and emits fluorescence that is captured by the Light Scanner HR 96 instrument. As temperature increases double-stranded DNA is converted to single-stranded, which dissociates LC Green from DNA, resulting in a decrease in fluorescence. Since melting of DNA is dependent on sequence and length, each amplicon has a unique melt profile. The data were analysed with LightScanner software (Idaho Technologies Inc.).

Kumba wildtype and heterozygotes adults were genotyped with oligonucleotides RA247 and

R248 (Appendix Table A1.2), whilst mNet wildtype and heterozygote adults and embryos were genotyped with RA1058 and RA1059. A Shh control was also amplified (RA748 and RA749) to ensure the integrity of mNet embryo gDNA.

2.9.4 Allelic Discrimination Assay (ADA)

ADA was used to genotype Kumba embryo DNA, as Zic2Ku/Ku _{homozygotes cannot be}

distinguished via HRMA. A TaqMan® Universal PCR Master Mix (Life Technologies; Cat. No. 4304437), along with TaqMan® MGB probes (Applied Biosystems) corresponding to the wildtype and Kumba (Ku) alleles of Zic2 were used to visualize the genotype of each embryo. The sequence of the probes are given in Table 2.13. The wildtype probes were modified at the 5’ end with a 6-FAM (6-carboxyfluorescein) tag and the mutant probes with VIC®. The 3’ end of each probe was modified with a non-fluorescent quencher and a minor groove binding moiety. The probes were used at half the recommended concentration (200 nM). All reactions were carried out in a final volume of 10 μL with ~30 ng of digested embryo DNA in the presence of 0.9 μM of oligonucleotides RA247 and RA248 (Appendix Table A1.2). Reactions were set up in 96-well Half- Skirted PCR Microplates (Axygen®; Cat. No. PCR-96-LP-AB-C) covered 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 normalized dye fluorescence (∆Rn) from the wildtype and mutant alleles as a function of cycle number. Based on this data the software called the sample as homozygous for either wildtype or mutant allele, or heterozygous with both alleles.

Table 2.12: Touchdown PCR cycling conditions.

Program: TD60 TD65 TD70

Initial

denaturation 94 °C 4 minutes 94 °C 4 minutes 94 °C 4 minutes Denature 94 °C 30 seconds X 29 94 °C 30 seconds X 19 92 °C 30 seconds X 20 Anneal 60 °C, decreasing by 0.5 °C per cycle, 30 seconds 65 °C, decreasing by 0.5 °C per cycle, 30 seconds 70 °C, decreasing by 0.5 °C per cycle, 30 seconds Extend 72 °C 30 seconds 72 °C 30 seconds 72 °C 1 minute Denature 94 °C 30 seconds X 19 94 °C 30 seconds X 29 92 °C 30 seconds X 20 Anneal 45 °C 30 seconds 55 °C 30 seconds 60 °C 30 seconds Extend 72 °C 30 seconds 72 °C 30 seconds 72 °C 1 minute

Polish 72 °C 7 minutes 72 °C 7 minutes 72 °C 7 minutes

Stop 4 °C Hold 4 °C Hold 4 °C Hold

Table 2.13: ADA probes for genotyping of Kumba mice.

Probe name 5’ Tag Probe sequence 3’ Quencher Zic2 WT 6-FAM CGA GGG CTG TGA CC MGBNFQ