Identification of an intestine-specific promoter and inducible expression of bacterial ?-galactosidase in mammalian cells by a lac operon system.

ABSTRACT: UNLABELLED: BACKGROUND:?-galactosidase has been widely used in animal husbandry to reduce anti-nutritional factors (such as ?-galactoside) in feed. Intestine-specific and substrate inducible expression of ?-galactosidase would be highly beneficial for transgenic animal production. METHODS:To achieve the intestine-specific and substrate inducible expression of ?-galactosidase, we first identified intestine-specific promoters by comparing the transcriptional activity and tissue specificity of four intestine-specific promoters from human intestinal fatty acid binding protein, rat intestinal fatty acid binding protein, human mucin-2 and human lysozyme. We made two chimeric constructs combining the promoter and enhancer of human mucin-2, rat intestinal trefoil factor and human sucrase-isomaltase. Then a modified lac operon system was constructed to investigate the induction of ?-galactosidase expression and enzyme activity by isopropyl ?-D-1-thiogalactopyranoside (IPTG) and an ?-galactosidase substrate, ?-lactose.We declared that the research carried out on human (Zhai Yafeng) was in compliance with the Helsinki Declaration, and experimental research on animals also followed internationally recognized guidelines. RESULTS:The activity of the human mucin-2 promoter was about 2 to 3 times higher than that of other intestine-specific promoters. In the lac operon system, the repressor significantly decreased (P < 0.05) luciferase activity by approximately 6.5-fold and reduced the percentage of cells expressing green fluorescent protein (GFP) by approximately 2-fold. In addition, the expression level of ?-galactosidase mRNA was decreased by 6-fold and ?-galactosidase activity was reduced by 8-fold. In line with our expectations, IPTG and ?-lactose supplementation reversed (P < 0.05) the inhibition and produced a 5-fold increase of luciferase activity, an 11-fold enhancement in the percentage of cells with GFP expression and an increase in ?-galactosidase mRNA abundance (by about 5-fold) and ?-galactosidase activity (by about 7-fold). CONCLUSIONS:We have successfully constructed a high specificity inducible lac operon system in an intestine-derived cell line, which could be of great value for gene therapy applications and transgenic animal production.

SUBMITTER: Ya-Feng Z 

PROVIDER: S-EPMC3527164 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature