INAPPROPRIATE SPLICING OF A CHIMERIC GENE CONTAINING A LARGE INTERNALEXON RESULTS IN EXON SKIPPING IN TRANSGENIC MICE

We generated transgenic mice containing a chimeric construct consistin g of the alpha-cardiac myosin heavy chain (alpha cMHC) promoter and th e human renin (hRen) gene in order to target hRen synthesis specifical ly to the heart, The construct consisted of three segments: (i) an alp ha cMHC DNA segment including 4.5 kb of 5' flanking DNA and an additio nal 1.1 kb of genomic DNA encompassing exons I-III (non-coding) and th e first two introns; (ii) a partial hRen cDNA consisting of exons I-VI ; and (iii) a hRen genomic segment containing exons VII through IX, th eir intervening introns, and 400 bp of 3' flanking DNA, This results i n the formation of a 909 bp internal fusion exon consisting of alpha c MHC, polylinker, and hRen sequences, Despite the presence of splice ac ceptor and donor sites bracketing this exon, transcription of this tra nsgene resulted in a major alternatively spliced mRNA lacking the exon and therefore a majority of the hRen coding sequence, Cloning and seq uencing of RT-PCR products from several heart samples from two indepen dent transgenic lines confirmed accurate and faithful splicing of alph a cMHC exon II to hRen exon VII thus bypassing the internal fusion exo n, All other exons (alpha cMHC exons I and II and hRen exons VII, VIII and IX) were appropriately spliced, These results are consistent with the hypothesis on exon definition which states that internal exons ha ve a size limitation, Moreover, the results demonstrate that transgene s present in the genome at independent insertion sites and in either a single copy or multiple copies can be subject to exon skipping, The i mplications for transgene design will be discussed.