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

Background: o-galactosidase has been widely used in animal husbandry to reduce anti-nutritional factors (such as o-galactoside) in feed. Intestine-specific and substrate inducible expression of a-galactosidase would be highly beneficial for transgenic animal production. Methods: To achieve the intes...

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Veröffentlicht in:畜牧与生物技术杂志:英文版 2013 (1), p.65-74
1. Verfasser: Yafeng Zhai Gang Shu Xiaotong Zhu Zhiqi Zhang Xiajing Lin Songbo Wang Lina WangYongliang Qingyan Jiang
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Sprache:eng
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Zusammenfassung:Background: o-galactosidase has been widely used in animal husbandry to reduce anti-nutritional factors (such as o-galactoside) in feed. Intestine-specific and substrate inducible expression of a-galactosidase would be highly beneficial for transgenic animal production. Methods: To achieve the intestine-specific and substrate inducible expression of o-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 o-galactosidase expression and enzyme activity by isopropyl p-D-]-thiogalactopyranoside (IPTG) and an a-galactosidase substrate, a-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/ac 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 o-galactosidase mRNA was decreased by 6-fold and a-galactosidase activity was reduced by 8-fold. in line with our expectations, IPTG and a-lactose supplementation reversed (P 〈 O.O5) 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 o-galactosidase mfiNA abundance (by about 5-fold) and o-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.
ISSN:1674-9782
2049-1891