4.2.1 In vitro fertilisation
IVF was performed by MRC Mammalian Genetics Unit (Harwell, Oxfordshire). Full details are provided in the Appendix (8.2). In brief, young sexually mature female mice were superovulated (ovulation was induced by administration of gonadotropin). Oocyte harvest, fertilisation and sperm dispersal dishes were prepared. Sperm was rapidly thawed and oocytes harvested from the
superovulated females. Sperm and oocytes were added to the prepared
fertilisation dish and incubated at 37C to allow fertilisation to occur. IVF
success was scored and 2-cell embryos were transferred into pseudopregnant females to mature.
4.2.2 Subjects and animal husbandry
Subjects obtained from the MRC Mammalian Genetics Unit, Harwell were housed and bred in the Behavioural Neurosciences Laboratory, School of Psychology, Cardiff University. Mice were group housed in cages of 2-5 subjects except where fighting or bullying necessitated single caging. Cages were environmentally enriched (with chew sticks and cardboard tubes) and standard laboratory food and water were available ad libitum. The light-dark cycle of the holding room was 12 hours with lights on at 07:00 and lights off at 19:00.
Experimental animals were regularly monitored for signs of ill health. Any mice showing signs of illness were immediately assessed by the Named Animal Care and Welfare Officer and, if necessary, the University’s vet. Only mice
showing no signs of ill health were used for breeding. If necessary, mice were withdrawn from breeding and treated or sacrificed, dependent upon the nature and presumed severity of the illness.
4.2.3 Breeding
Mice were used for breeding from approximately one to three months of age. Females were introduced to the male’s home cage. If this was not possible then
both male and female animals were introduced to a new cage. Tubing was removed from cages to facilitate breeding. Breeding cages ranged from one to four females with one male and breeding pairs/ harems were left undisturbed for one week. After this period females were checked for signs of pregnancy or a semen plug and left with the male for a further week if required. If any
females were not pregnant following two weeks exposure to the male, they were removed from the breeding programme. If the male (by process of elimination) was not thought to be fertile, it was removed from the breeding programme.
4.2.4 Tail biopsy
See 2.7; Tail biopsy in General Materials and Methods
4.2.5 Genotyping
Genotyping methods followed those described in 2.2 used for mutation discovery but for substituting LC Green in the PCR master mix for double distilled H20. The LightScanner stage was also not performed as it was not
necessary. PCRs were cleaned as per protocol and sequencing was performed.
4.2.6 Culling
Mice no longer required for the breeding programme were culled according to the protocol outlined in 2.8 Culling protocol in General Materials and Methods.
4.2.7 Linkage panel
The Mouse LD (low density) Linkage Panel (Illumina, Inc) was used to genotype 377 SNPs in mice carrying the Zfp804a mutations (see 2.3.1 and figure 2.2 for methods). The SNPs were pre-selected by Illumina as they are known to be polymorphic between the 10 most common strains of mouse. Coverage is approximately four SNPs every 27Mb, with at least one of these four SNPs being informative for crosses involving the C57Bl/6J strain. The Panel uses the GoldenGate Gentoyping Assay protocol (Illumina, Inc). Full details of the protocol are provided on Illumina’s website (www.illumina.com).
Genomic DNA (gDNA) was normalised to 50 – 60 ng/µl (250ng minimum in total required). DNA was then ‘activated’ by treating it with streptavidin-biotin
to prepare the gDNA for binding to paramagnetic particles. Assay
oligonucleotides, hybridisation buffer and the paramagnetic particles were then added to the DNA for the hybridisation stage. Three oligonucleotides are provided for each SNP: two oligos are allele-specific and the third is designed to anneal several bases downstream from the allele that ligates to the extended allele-specific oligos in the next phase following several wash steps to remove
excess and mis-hybridised oligos. Following this, PCR is performed on the samples using three universal PCR primers. Single-stranded DNAs are hybridised to their complement bead. These products were then hybridised onto the BeadChip and dried to isolate the assay products for individual
analysis. After the products were hybridised, the SNPs were analysed using the BeadArray Reader (which analyses fluorescence signal). This is then analysed by software for automated genotype clustering and calling. The GenomeStudio Genotyping Module uses a clustering algorithm to call genotypes, depending on the threshold set by the user. Further details of the software are contained in the Technical Note available online (www.illumina.com). Further analysis was performed in Microsoft Excel.
Figures 4.1 and 4.2 below show the layout of the results from the Mouse LD Linkage Panel. Figure 4.1 shows the SNP, the C57Bl/6J genotype, the chromosome, genomic position and quality score (1 is the highest). Columns J to P shown represent individual mice. Figure 4.2 is a different view of the same spreadsheet demonstrating how the estimated percentage C57Bl/6J was calculated (for the formula used, see figure 4.3).
Figure 4.1 (above): Layout of results from G4i linkage panel showing the SNP, chromosome, position, C57Bl/6J genotype and genotypes of individual mice (per column). Each row represents a SNP and each column represents a mouse (J-P).
Figure 4.2: Screenshot of the spreadsheet used to calculate % C57Bl/6J of the mice, demonstrating how the % congenicity was arrived at.
The percent congenicity of the mice (or % C57Bl/6J) was estimated by the proportion of successfully genotyped alleles that were of C57Bl/6J origin (see Figure 4.3). The equation below illustrates how the percent C57Bl/6J
congenicity for each mouse was then calculated, where N=number of SNPs successfully genotyped, het= number of SNPs heterozygous (1 allele C57Bl/6J, 1 allele BALB/c or C3H/HeJ), hom=number of SNPs homozygous (both alleles non-C57Bl/6J),
Figure 4.3: Equation to demonstrate the calculation of percentage congenicity for C57Bl/6J
Due to comparatively low numbers of markers used, the data were not suitable to be analysed by programs such as PLINK (a whole-genome data analysis toolset).