Figure 7. Unique alternative splice variants in the AChE del E5+6 knockout mouse.

a) Maps of the AChE gene with alternative splice variants for the WT and AChE del E5+6 knockout show that in the knockout exons 5 and 6 are deleted and replaced by a short sequence used in the generation of the knockout (orange bar). The predicted mature protein produced by the knockout comes from AChE_R cDNA corresponding to the absence of splicing after exon 4, retaining 3′ genomic sequence. Two alternative splice variants, named variant 2 and 3, were identified in the AChE del E5+6^−/− knockout, and are shown. Note that there are two potential signals for poly-adenylation.

b) Semi-quantitative RT-PCR with s3 and UT1 primers. In WT, s3-UT1 amplified a single PCR product of 507 bp (3+4+6) corresponding to the AChE_T mRNA isoform. As expected, this band is absent in AChE del 5+6 knockout, but we detected two bands of 683 bp and 255 bp. The 683 bp band reflects the predicted unspliced AChE_R form (3+4+R), whereas the 255 bp product corresponds to splice variant 2. Quantification of the bands shows that AChE_R represents ~20% of WT mRNA levels (set as 100%), while variant 2 represented only ~3%. c) Semi-quantitative RT-PCR with s3 and UT2 primers. We found a single band of 1737 bp in the WT (AChE_T) and a novel band of 1159 bp (variant 3, fusion of AChE with UfSP1) in the AChE del5+6 knockout.

c) Semi-quantitative RT-PCR with s3 and UT2 primers. We found a single band of 1737 bp in the WT (AChET) and a novel band of 1159 bp (variant 3, fusion of AChE with UfSP1) in the AChE del5+6 knockout.

d) The exact locations of splice acceptor sites found in the AChE del E5+6 knockout by RT-PCR and sequencing are shown. Potential translated amino acid sequence is shown above the DNA sequence. Numbering comes from the reference sequence (AF312033.1) (Wilson et al., 2001).