G. Woerfel et A. Bindereif, In vitro selection of exonic splicing enhancer sequences: identification of novel CD44 enhancers, NUCL ACID R, 29(15), 2001, pp. 3204-3211
We have developed an in vitro selection procedure that allows the identific
ation and isolation of functional splicing enhancer sequences from any cDNA
. It is based on the enhancement of general splicing activity of a pre-mRNA
reporter derived from the Drosophila dsx gene. Short DNase I fragments are
cloned into a cassette in the second exon of the reporter construct, repla
cing the natural dsx enhancer. After splicing and reverse transcription-PCR
, fragments are recovered from the mRNA product. Applying this selection to
the CD44 gene, which undergoes extensive alternative splicing processes, w
e have identified several novel exonic enhancers. Two of them, which reside
in CD44 variable exon 6, were further characterized by mutational analysis
and confirmed to function within their natural CD44 context.