Genotyping methods
Several Southern blot and PCR strategies were exploited in order to determine the genotype of the Capn2 locus. Southern blotting was carried out using the digoxigenin (DIG) non-radioactive system (Roche). In most cases, membranes were blotted with BamHI-digested genomic DNA and hybridized with a DIG-labeled 823 bp exon 4-containing BamHI-HindIII fragment located immediately upstream of the short arm of homology (Figure 1). A 681 bp PstI-XbaI fragment from the PGK-Neo cassette was also used to probe Southern blots in order to verify a single integration event in targeted clones.
Genotyping was also carried out by PCR analysis of genomic DNA. The sequences of all oligonucleotide primers are listed in Table 1. A single-step PCR strategy was sufficient for genotyping ES cells or biopsies from post-implantation embryos and weanlings (Figure 1). A 2,748 bp segment of the wild-type allele and a 2,711 bp segment of the mutant allele were amplified in separate reactions using a common (intron 4) sense primer, located outside the short arm of homology, and distinct antisense primers which hybridized to either wild-type (exon 7) or mutant (PGK-Neo) sequence (Table I). The thermocycling parameters included a five minute initial denaturation step at 95°C, 30 cycles of one minute denaturation at 95°C, one minute annealing at 56°C, and one minute extension at 72°C, with a ten minute final extension step.
Table 1  Oligonucleotide primers used to genotype the Capn2 locus
Allele  Primer  Location  Oligonucleotide Sequence
Single-Step
Both  Sense  Intron 4  5'-GGGCCCCCATTGCCTCTTAGC-3'
Wild-type  Antisense  Exon 7  5'- GGATTCCTGATGCGGATCAATTTCTGC-3'
Mutant  Antisense  PGK-Neo  5'-CCTCGAAGTCGAGGTCGATCC-3'
Nested PCR
Wild-Type (Diagnostic)  Sense #1  Intron 6  5'-CAACATCATAAGCAACGGAGAACGC-3'
Sense #2  Intron 6  5'-GCCTGTGACAGAAGTACCACCAG-3'
Antisense #1  Intron 7  5'-CTCCTCGGCCCTCCCTGTAG-3'
Antisense #2  Exon 7  5'-GGATTCCTGATGCGGATCAATTTCTGC-3'
Mutant (Diagnostic)  Sense #1  Intron 6  5'-CAACATCATAAGCAACGGAGAACGC-3'
Sense #2  Intron 6  5'-GCCTGTGACAGAAGTACCACCAG-3'
Antisense #1  PGK-Neo  5'-CCTACCCGGTAGAATTGACCTGC-3'
Antisense #2  PGK-Neo  5'-GACCTGCAGGGGCCCTC-3'
Both (Internal Control)  Sense #1  Intron 4  5-GGGATGAAGGCTCCCTGTTGC-3'
Sense #2  Exon 5  5'-GGGGGTGCCACCACGGAA -3'
Antisense #1  Intron 5  5'-CCCCTAGAGGACTATTGCCTGG-3'
Antisense #2  Intron 5  5'-GATGCACCCATGCCTTAAGGAGC-3' Due to the limited amount of genetic material available in pre-implantation embryos, a nested PCR strategy was developed to yield reliable genotyping information (Figure 4). Whole embryos were first digested in 20 μL of proteinase K buffer (see below). The lysate was then divided in two, with half (10 μL) being used in the amplification of the wild-type allele and the remaining 10 μL in the amplification of the mutant allele.
The first reaction in the nested PCR amplification of the wild-type allele was carried out in a final reaction volume of 50 μL, using an intron 6 sense primer and an antisense primer located in intron 7. Two μL of the first reaction were used as template in the second PCR amplification using another intron 6 sense primer and an antisense primer in exon 7. The nested amplification of the mutant allele was carried out similarly. The first primer pair consisted of the intron 6 sense primer and an antisense primer in the PGK-Neo cassette. The nested primer pair was comprised of the second intron 6 sense primer and an antisense primer also located within the PGK-Neo sequence. It should be noted that amplification of both sequences involved the same sense primers in both steps of the nested PCR strategy. In addition, the two sets of reactions included a common internal control designed to amplify a genomic region within the short arm of the targeting vector that is preserved in both the wild-type and mutant alleles. The first primer pair of the control PCR was made up of an intron 4 sense primer and an intron 5 antisense primer. The second primer pair comprised an exon 5 sense primer and a nested intron 5 antisense primer. The final products were 213 bp for the control PCR, 429 bp for the wild-type PCR, and 389 bp for the mutant PCR. All reactions were carried out using identical PCR conditions entailing an initial five minute denaturation at 95°C, 35 cycles of one minute denaturation at 95°C, one minute annealing at 56°C, and one minute extension at 72°C, with a final extension step of ten minutes.