SYNTHESIS OF A LACI GENE ANALOG WITH REDUCED CPG CONTENT

A lacI gene analogue with reduced CpG content has been synthesized. Co don usage in the lacI gene was manipulated to remove most CpG sites (8 2/95; 86%) while maintaining wild-type amino acid sequence. The double -stranded gene sequence was synthesized using standard beta-cyanoethyl phosphoramidite chemistry and subsequently cloned into pBR322. Bacter ial promoter sequences with different levels of activity were attached upstream of the modified coding region to study its expression in E. coli. Production of lacI protein was confirmed in a lacI(-) E. coli st rain by Western blot analysis and by measuring repression of the lacZ gene with the chromogenic lacZ indicator, -bromo-4-chloro-3-indolyl-be ta-D-galactopyranoside (X-gal). The modified lad gene construct can be used as a genetic target in cultured mammalian cells or in transgenic animals to avoid high levels of background mutation associated with m ethylated CpG sequences. The construction scheme described here provid es a general approach to remove CpG sequences from gene constructs whe n methylation is undesirable.