Identification of Alternatively Spliced Variants from Opioid Receptor Genes

Alternative splicing is commonly used in eucaryotic gene regulation. A single mouse μ opioid receptor gene (Oprm ) can generate 15 spliced variants by alternative splicing of its 14 exons (1 –6 ). Furthermore, the region-specific expression of the splice variants suggests an important role of alternative splicing in regulating gene functions (4 –8 ). Genomic sequences from various species are available from many genome projects, but it is still challenging to directly identify splice variants from a given gene sequence through sequence analysis, particularly for those with cryptic splice sites. Since some of the variants are expressed at a lower level, traditional library screening often fails to identify such variants. Rapid development of polymerase chain reaction (PCR) technology provides a powerful tool of isolating low-level splice variants with great sensitivity and specificity. There are several common patterns of alternative splicing including exon skipping, intron retention, and exon recruiting. This chapter describes three similar, but distinct approaches of identifying the potential low-level splice variants: 1) a modified rapid amplification of cDNA 5′- end (5′RACE) approach used to isolate the variants with new exons at the 5′-end; 2) a modified 3′ RACE approach used to obtain the variants with new exons at the 3′-end; and 3) an internal exon-scanning reverse transcriptase (RT)-PCR approach used to identify the variants with new exons from the introns or with exon skipping.