Figure 5

Molecular analysis of mCat-1 mutations. (a) Junction fragment analysis of retroviral resistant clones. Southern blot of retroviral infection resistant clones isolated from different subpools of the gene trap library. The clones isolated from different subpools have different HindIII proviral-host junction fragments and are thus independent mutants, as expected. The clones isolated form the same pools share a common host-proviral junction fragment and thus appear to be daughter clones in all cases. The probe is the ClaI fragment from the SA-βgeo cassette. LTR, long terminal repeat. (b) Insertion sites of gene-trap virus in mCat-1. Part 1 shows the structure of the 5' end of mCat-1; the initiating ATG codon is in exon 3. The proviral-host junction at the end of 5' LTR was sequenced by Splinkerette PCR. The location of the retroviral insertions is shown by arrows. Nucleotide positions are from National Center for Biotechnology Information (NCBI) mouse build 35. The pools that correspond to V1 to V5 are shown in brackets. The structure of the fusion transcripts for the insertions in introns 1 and 2 are also shown in parts 2 and 3, respectively. (c) Homozygosity analysis of mCat-1 in retroviral resistant clones. Southern blot with an mCat-1 probe. Absence of the wild-type fragment in V1, V2, V4, and V5 indicates that the viral insertions are homozygous. The presence of the wild-type fragment in the V3 clone reveals that in this clone the insertion is heterozygous. (d) Expression of wild-type and fusion transcripts in gene-trap mutants. As shown in part 1, reverse transcription (RT)-polymerase chain reaction (PCR) with primers for mCat-1 exon 1 and lacZ detected fusion transcripts of the expected size. Fusion transcripts of 238 base pairs (bp) were amplified from clones V1, V2 and V3, whereas transcripts of 328 bp were detected from clones V4 and V5. The wild-type exon 1 to 3 transcript was only amplified from Blm-deficient ES cells. β-Actin (Actb) served as a positive control for RT-PCR. As shown in part 2, RT-PCR with primers for mCat-1 exon 4 to 7 and exon 8 to 12 detected trace transcript levels of mCat-1 from mutant clones except V1. The 403 bp product detected by RT-PCR for exon 4 to 7 primers in the V3 clone was sequenced and found to have an aberrant splice, as illustrated in part 3.