Figure 4.

The gene-trapped Notch3 allele is a null allele. Random insertion of the lacZ gene trap was suspected to be in the vicinity of exons 16-17 of Notch3. (A) The location of a 2.1-kb amplicon resulting from a forward primer in exon 16 and a reverse primer in exon 17. (B) Insertion of the lacZ gene trap would generate an amplicon too large for conventional PCR using these primers; presence of the gene trap can also be confirmed using gene trap-specific primers. (C) The expected results are found using DNA from wild-type mice, homozygous gene-trapped mice, or heterozygous mice. (D) To exclude the possibility of alternate transcripts that might skip the gene-trap insert, PCR was performed in the 3′ region of the transcript (spanning exons 32 and 33), revealing that no detectable transcripts could be found. (E) An Ab recognizing the C-terminal (intracellular) domain of Notch3 was used to probe extracts from N3^lacZ/lacZ or wild-type lineage-negative thymocytes. No detectable protein could be found. Together these experiments confirm that the gene-trapped allele is a null allele.