An improved beta-galactosidase reporter gene.
The coding sequence for the E. coli beta-galactosidase gene was codon-optimised for expression in mammalian cells. When expressed in mammalian cells the codon-optimised gene results in the expression of beta-galactosidase at levels 15-fold higher than those resulting from an analogous construct containing the native E. coli gene sequence. RNA analysis suggests the enhancement of beta-galactosidase expression is due both to enhanced transcript stability and increased translational efficiency. When used in a lentiviral construct the codon-optimised gene results in an approximately five-fold increase in apparent titre, as determined by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining, in comparison to an analogous construct containing the native E. coli gene. Southern blot analysis shows this is due to an increased efficiency of detection of transduced cells. In addition, codon-optimisation results in the elimination of several cryptic splice acceptor sites that are present in the native E. coli gene sequence. In a lentiviral vector containing a 5' splice donor the use of the codon-optimised gene in place of the native E. coli beta-galactosidase gene resulted in increased amounts of un-spliced, full-length genomic RNA. Therefore, as a marker/reporter gene in mammalian cells the codon-optimised beta-galactosidase gene has a number of advantages over the native E. coli gene sequence. A variant of the codon-optimised beta-galactosidase gene sequence that includes an effective nuclear localisation signal was also made.
Anson
D S
DS
Department of Chemical Pathology, Women's and Children's Hospital, 72 King William Road, North Adelaide, SA 5006, Australia. donald.anson@adelaide.edu.au
Limberis
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018929:Cell Culture Techniques; D002460:Cell Line; D016384:Consensus Sequence; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D007763:Lac Operon; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics* / Q000378:metabolism
D001616:beta-Galactosidase