Table 2  Genotype distribution of offspring derived from Capn2 transgenic mice.
Cross  (N)  Age  Genotype
+/+  +/-  -/-  ND*
(+/-)♀ × (+/-)♂  199  3 weeks  23  176  0  0
(+/+)♀ × (+/-)♂  122  3 weeks  50  72  0  0
(+/-)♀ × (+/+)♂  103  3 weeks  28  75  0  0
(+/-)♀ × (+/-)♂  9  E18.5  0  7  0  2
(+/-)♀ × (+/-)♂  5  E17.5  0  5  0  0
(+/-)♀ × (+/-)♂  9  E14.5  2  7  0  0
(+/-)♀ × (+/-)♂  10  E11.5  3  7  0  0
(+/-)♀ × (+/-)♂  7  E10.5  2  5  0  0
(+/-)♀ × (+/-)♂  46  E3.5  7  39  0  0
(+/-)♀ × (+/-)♂  48  E2.5  8  34  2  4
* ND, not determined
Figure 4  Nested PCR strategy for genotyping of pre-implantation embryos. Due to the scarcity of extractable genetic material, a nested PCR strategy was developed in order to genotype pre-implantation embryos. Separate reactions were used to amplify a 429 bp fragment from the wild-type allele and a 389 bp segment from the mutant allele, both spanning the 3' end of the short (upstream) arm of the targeting vector. In both reactions, a 213 bp sequence located within the short arm was co-amplified with the 'diagnostic' products as an internal control. The same sense primers were used to amplify 'diagnostic' sequences in both reactions, whereas the antisense primers were allele-specific. The primers, represented by triangles, are depicted in two (nested) sets for each of the three reactions. Exons are represented by open, vertical rectangles, the PGK-Neo cassette by an open, horizontal rectangle, while thin vertical lines denote the boundaries of the short arm and the 5' end of the long (downstream) arm. A grey, horizontal rectangle delineates the segment of the wild-type allele that is replaced by the PGK-Neo cassette in the mutant allele.
Figure 5  Genotyping of pre-implantation embryos. A nested PCR strategy was used to genotype embryos prior to implantation as described in Figure 4. Capn2+/- mice were mated and the date of fertilization established by the appearance of vaginal plugs. Blastocyst (E3.5) or 8-cell embryos (E2.5) were flushed from the oviducts and then digested with proteinase K. In separate reactions segments found exclusively in either the wild-type or mutant alleles were co-amplified with an internal control sequence, located in the short (upstream) arm of the targeting vector, which is found in both alleles. The final products were 429 bp for the wild-type allele, 389 bp for the mutant allele, and 213 bp for the internal control. (A) A representative example of the genotyping of blastocyst stage embryos. Embryos #1, 2, 4, 5, and 6 were Capn2+/- whereas embryos #3 and #7 were Capn2+/+, denoted by the absence of the 389 bp mutant signal. (B) An example of the genotyping of 8-cell embryos is shown. Embryos #1, 2, 3, 5, and 6 were Capn2+/- while embryo #4 was Capn2-/-, marked by the absence of the 429 bp wild-type signal. (M) denotes the molecular weight marker. The genotyping results for weanlings, post-implantation, and pre-implantation embryos are shown in Table 2.