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. 2011 Nov 23;6(11):e27940. doi: 10.1371/journal.pone.0027940

Figure 3. Defective splicing due to a deletion in intron 5 of canine LHX3 demonstrated by exon trapping.

Figure 3

(A) Map of part of the plasmids used for transfection of COS-7 cells. The open and closed boxes represent exons 5 and 6 of canine LHX3, as indicated. The hatched boxes represent chimeric exons that consist of HIV-1 tat and rabbit HBB2 gene sequences. The double lines indicate vector sequences of pSPL3b [50]. The recombinant DNA is transcribed from the SV40 promoter at the indicated position. The map is not drawn to scale. The horizontal arrows indicate the positions of the PCR primer sets I and II that were used on cDNA derived from the cells. Primer set II was adapted to bisulfite treatment of the RNA. The vertical arrow points out intron 5, consisting of 75 bp in the control and 68 bp in the mutant construct. (B,C) Eukaryotic COS-7 cells were transfected with recombinant plasmids containing the control or the mutant intron 5 of canine LHX3. After 2 days, RNA was isolated and analyzed by RT-PCR. The PCR products were analyzed by agarose gel electrophoresis in the presence of ethidium-bromide. The structure of the fragments was obtained by DNA sequence analysis of bands excised from the gels. These structures are indicated by drawings as in (A). (B) The 7 bp deletion leads to exon 5 skipping in a proportion of the transcripts. The products were analyzed with primer set I. Lane 1: size marker = 100 bp ladder, * indicates the 500 bp fragment; lane 2: products derived from cells transfected with plasmids containing intron 5 of 75 bp; lane 3: intron 5 of 68 bp; lanes 4 and 5: as lanes 2 and 3, respectively, except that reverse transcriptase was omitted to exclude contamination of RNA substrate with plasmid DNA; lane 6: transfection with vector pSPL3b without insert; lane 7: no transfection. Primer set I does not amplify fragments containing intron 5 due to the high cytosine content of the intron. (C) The 7 bp deletion leads to retention of intron 5 in a proportion of the transcripts. The RNA was treated with bisulfite and analyzed with primer set II. Lanes 1 and 4: products derived from cells transfected with plasmids containing intron 5 of 75 bp; lanes 2 and 5: products derived from cells transfected with plasmids containing intron 5 of 68 bp; lane 6: transfection with vector pSPL3b without insert; lane 3: size marker = 100 bp ladder, * indicates the 500 bp fragment. Lanes 4 and 5: omission of reverse transcriptase to exclude contamination of RNA substrate with plasmid DNA.