Table 2.
Polymerase Chain Reaction–Based Genotyping of CD3 Alleles Confirms Mendelian Inheritance of the Wild-Type and Knockout Genes
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No. of F3 mice/genotype |
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|---|---|---|---|---|---|---|---|---|---|
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F2 breeding pair |
Expected Mendelian distribution |
Observed by PCR |
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| Parent 1 | Parent 2 | WT | HT | KO | WT | HT | KO | ||
| PCR CD3ɛ genotype | |||||||||
| 1 | HT | HT | 1.5 | 3 | 1.5 | 3 | 1 | 2 | |
| 2 | HT | WT | 3.5 | 3.5 | 0 | 4 | 3 | 0 | |
| 3 | HT | KO | 0 | 2 | 2 | 0 | 1 | 3 | |
| 4 | HT | HT | 1.25 | 2.5 | 1.25 | 2 | 2 | 1 | |
| 5 | HT | WT | 3.5 | 3.5 | 0 | 3 | 4 | 0 | |
| Total number of mice | 9.75 | 14.5 | 4.75 | 12 | 11 | 6 | p = 0.4289 | ||
| PCR CD3ζ genotype | |||||||||
| 1 | HT | KO | 0 | 3 | 3 | 0 | 4 | 2 | |
| 2 | HT | KO | 0 | 3.5 | 3.5 | 0 | 4 | 3 | |
| 3 | HT | KO | 0 | 2 | 2 | 0 | 1 | 3 | |
| 4 | KO | WT | 0 | 5 | 0 | 0 | 5 | 0 | |
| 5 | HT | HT | 1.75 | 3.5 | 1.75 | 1 | 3 | 3 | |
| Total number of mice | 1.75 | 17 | 10.25 | 1 | 17 | 11 | p = 0.8285 | ||
F2 offspring resulting from intercrossed F1 mice (CD3γδɛζ−/− × B6) were genotyped for CD3ɛ and CD3ζ genes by PCR. Subsequently, five breeding pairs were arranged that consisted of F2 parents with heterogeneous genotypes for both genes. The table summarizes the PCR genotypes of the F2 parents, and the numbers of expected versus PCR-observed genotypes among the F3 offspring. The chi-square test showed that the numbers of F3 mice found by PCR for each possible CD3ɛ and CD3ζ genotype were not significantly different from their expected frequencies based on a Mendelian distribution of the CD3ɛ and CD3ζ WT and KO alleles (p > 0.05).
PCR, polymerase chain reaction; HT, heterozygote.