Cryptic splice site in the complementary DNA of glucocerebrosidase causes inefficient expression.
The low levels of human lysosomal glucocerebrosidase activity expressed in transiently transfected Chinese hamster ovary (CHO) cells were investigated. Reverse transcription PCR (RT-PCR) demonstrated that a significant portion of the transcribed RNA was misspliced owing to the presence of a cryptic splice site in the complementary DNA (cDNA). Missplicing results in the deletion of 179 bp of coding sequence and a premature stop codon. A repaired cDNA was constructed abolishing the splice site without changing the amino acid sequence. The level of glucocerebrosidase expression was increased sixfold. These data demonstrate that for maximum expression of any cDNA construct, the transcription products should be examined.
Bukovac
Scott W
SW
Research Institute, Hospital for Sick Children, Toronto, Ont., Canada M5G 1X8.
Bagshaw
Richard D
RD
Rigat
Brigitte A
BA
Callahan
John W
JW
Clarke
Joe T R
JT
Mahuran
Don J
DJ
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004252:DNA Mutational Analysis; D018076:DNA, Complementary; D005776:Gaucher Disease / Q000209:etiology / Q000235:genetics; D005962:Glucosylceramidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D022821:RNA Splice Sites; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018076:DNA, Complementary; D022821:RNA Splice Sites; D005962:Glucosylceramidase