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

Ya-Feng Zhai, Gang Shu, Xiao-Tong Zhu, Zhi-Qi Zhang, Xia-Jing Lin, Song-Bo Wang, Li-Na Wang, Yong-Liang Zhang, Qing-Yan, Qing-Yan Jiang

Description

α-galactosidase has been widely used in animal husbandry to reduce anti-nutritional factors (such as α-galactoside) in feed. Intestine-specific and substrate inducible expression of α-galactosidase would be highly beneficial for transgenic animal production. Methods To achieve the intestine-specific and substrate inducible expression of α-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 α-galactosidase expression and enzyme activity by isopropyl β-D-1-thiogalactopyranoside (IPTG) and an α-galactosidase substrate, α-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 α-galactosidase mRNA was decreased by 6-fold and α-galactosidase activity was reduced by 8-fold. In line with our expectations, IPTG and α-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 α-galactosidase mRNA abundance (by about 5-fold) and α-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.

Sujets

• Lac operon

Informations

• Publié par: biomed
• Publié le: 01 janvier 2012
• Nombre de lectures: 169
• Langue: English

Extrait

YaFenget al. Journal of Animal Science and Biotechnology2012,3:32 http://www.jasbsci.com/content/3/1/32

JOURNAL OF ANIMAL SCIENCE AND BIOTECHNOLOGY

R E S E A R C HOpen Access Identification of an intestinespecific promoter and inducible expression of bacterial αgalactosidase in mammalian cells by alac operon system † † Zhai YaFeng , Shu Gang , Zhu XiaoTong, Zhang ZhiQi, Lin XiaJing, Wang SongBo, Wang LiNa, * Zhang YongLiang and Jiang QingYan

Abstract Background:αgalactosidase has been widely used in animal husbandry to reduce antinutritional factors (such as αgalactoside) in feed. Intestinespecific and substrate inducible expression ofαgalactosidase would be highly beneficial for transgenic animal production. Methods:To achieve the intestinespecific and substrate inducible expression ofα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 modifiedlacoperon system was constructed to investigate the induction ofαgalactosidase expression and enzyme activity by isopropyl βD1thiogalactopyranoside (IPTG) and anαgalactosidase substrate,α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 thelacoperon 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 ofαgalactosidase mRNA was decreased by 6fold and αgalactosidase activity was reduced by 8fold. In line with our expectations, IPTG andα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 inαgalactosidase mRNA abundance (by about 5fold) andαgalactosidase activity (by about 7fold). Conclusions:We have successfully constructed a high specificity induciblelacoperon system in an intestinederived cell line, which could be of great value for gene therapy applications and transgenic animal production. Keywords:αgalactosidase, Inducible expression, Intestinespecific promoters,Lacoperon

* Correspondence: [e-mail] † Equal contributors College of Animal Science, South China Agricultural University, Guangzhou 510642, China

© 2012 YaFeng et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.