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Ya-Feng Z, Gang S, Xiao-Tong Z, et al. Identification of an intestine-specific promoter and inducible expression of bacterial α-galactosidase in mammalian cells by a lac operon system. J Anim Sci Biotechnol. 2012;3(1):32doi: 10.1186/2049-1891-3-32.
Ya-Feng, Z., Gang, S., Xiao-Tong, Z., Zhi-Qi, Z., Xia-Jing, L., Song-Bo, W., Li-Na, W., Yong-Liang, Z., & Qing-Yan, J. (2012). Identification of an intestine-specific promoter and inducible expression of bacterial α-galactosidase in mammalian cells by a lac operon system. Journal of animal science and biotechnology, 3(1), 32. https://doi.org/10.1186/2049-1891-3-32
Ya-Feng, Zhai, et al. "Identification of an intestine-specific promoter and inducible expression of bacterial α-galactosidase in mammalian cells by a lac operon system." Journal of animal science and biotechnology vol. 3,1 (2012): 32. doi: https://doi.org/10.1186/2049-1891-3-32
Ya-Feng Z, Gang S, Xiao-Tong Z, Zhi-Qi Z, Xia-Jing L, Song-Bo W, Li-Na W, Yong-Liang Z, Qing-Yan J. Identification of an intestine-specific promoter and inducible expression of bacterial α-galactosidase in mammalian cells by a lac operon system. J Anim Sci Biotechnol. 2012 Oct 30;3(1):32. doi: 10.1186/2049-1891-3-32. PMID: 23111091; PMCID: PMC3527164.
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J Anim Sci Biotechnol. 2012 Oct 30;3(1):32. doi: 10.1186/2049-1891-3-32.

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

Journal of animal science and biotechnology

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

Affiliations

  1. College of Animal Science, South China Agricultural University, Guangzhou 510642, China. [email protected].

PMID: 23111091 PMCID: PMC3527164 DOI: 10.1186/2049-1891-3-32

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 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.

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