Cre-loxP system as a versatile tool for conferring increased levels of tissue-specific gene expression from a weak promoter.

@article{citeulike:2309334, abstract = {Attempts to image reporter gene expression driven by weak promoters are often hampered by the poor transcriptional activity of such promoters. Most tissue-specific promoters are weak compared with stronger but constitutively expressing viral promoters. In this study, we validated methods of enhancing the transcriptional activity of weak promoters using a Cre-loxP system in vitro and in vivo. We constructed a tester vector, pCTL, which carries a strong systemic cytomegalovirus enhancer/ chicken beta-actin promoter (CAG), loxP-flanked CAT, and firefly luciferase (luc) cDNAs. Herpes simplex virus-thymidine kinase (HSV-tk) promoter was used as a weak and systemic promoter and ligated to Cre for construction of pTC. Luc activity was higher (about 10-fold enhancement) in co-transfected (with pCTL and pTC) than in singly (with HSV-tk promoter-driven luc expression vector pTL) transfected NIH3T3 cells. In vivo electroporation-mediated gene delivery of both pCTL and pTC into murine oviductal epithelium yielded results (about 16-fold enhancement) similar to those obtained with in vitro-transfected NIH3T3 cells. To evaluate tissue-specific enhancement of gene expression, podocyte (glomerular visceral epithelial cell)-specific nephrin promoter was ligated to the Cre gene or luc cDNA to create pNC and pNL, respectively. We achieved 2.4-fold improvement of luc gene expression in the mouse kidney in vivo when pCTL and pNC were co-transfected via the tail vein via the lipoplex method. The combination of a weak tissue-specific promoter with the Cre-loxP system could thus be used to enhance the strength of tissue-specific promoters in vitro and in vivo. Mol. Reprod. Dev. (c) 2007 Wiley-Liss, Inc.}, address = {Department of Surgery, School of Medicine, National Defense Medical College, 3‐2 Namiki, Tokorozawa, Saitama, Japan.}, author = {Nakamura, Shingo and Watanabe, Satoshi and Ohtsuka, Masato and Maehara, Tadaaki and Ishihara, Masayuki and Yokomine, Takaaki and Sato, Masahiro }, citeulike-article-id = {2309334}, doi = {10.1002/mrd.20847}, issn = {1040-452X}, journal = {Mol Reprod Dev}, keywords = {plasmid}, month = {December}, posted-at = {2008-01-31 02:15:04}, priority = {2}, title = {Cre-loxP system as a versatile tool for conferring increased levels of tissue-specific gene expression from a weak promoter.}, url = {http://dx.doi.org/10.1002/mrd.20847}, year = {2007} }

TY - JOUR ID - citeulike:2309334 L3 - citeulike-article-id:2309334 TI - Cre-loxP system as a versatile tool for conferring increased levels of tissue-specific gene expression from a weak promoter. JF - Mol Reprod Dev AD - Department of Surgery, School of Medicine, National Defense Medical College, 3‐2 Namiki, Tokorozawa, Saitama, Japan. SN - 1040-452X N2 - Attempts to image reporter gene expression driven by weak promoters are often hampered by the poor transcriptional activity of such promoters. Most tissue-specific promoters are weak compared with stronger but constitutively expressing viral promoters. In this study, we validated methods of enhancing the transcriptional activity of weak promoters using a Cre-loxP system in vitro and in vivo. We constructed a tester vector, pCTL, which carries a strong systemic cytomegalovirus enhancer/ chicken beta-actin promoter (CAG), loxP-flanked CAT, and firefly luciferase (luc) cDNAs. Herpes simplex virus-thymidine kinase (HSV-tk) promoter was used as a weak and systemic promoter and ligated to Cre for construction of pTC. Luc activity was higher (about 10-fold enhancement) in co-transfected (with pCTL and pTC) than in singly (with HSV-tk promoter-driven luc expression vector pTL) transfected NIH3T3 cells. In vivo electroporation-mediated gene delivery of both pCTL and pTC into murine oviductal epithelium yielded results (about 16-fold enhancement) similar to those obtained with in vitro-transfected NIH3T3 cells. To evaluate tissue-specific enhancement of gene expression, podocyte (glomerular visceral epithelial cell)-specific nephrin promoter was ligated to the Cre gene or luc cDNA to create pNC and pNL, respectively. We achieved 2.4-fold improvement of luc gene expression in the mouse kidney in vivo when pCTL and pNC were co-transfected via the tail vein via the lipoplex method. The combination of a weak tissue-specific promoter with the Cre-loxP system could thus be used to enhance the strength of tissue-specific promoters in vitro and in vivo. Mol. Reprod. Dev. (c) 2007 Wiley-Liss, Inc. KW - plasmid AU - Nakamura, Shingo AU - Watanabe, Satoshi AU - Ohtsuka, Masato AU - Maehara, Tadaaki AU - Ishihara, Masayuki AU - Yokomine, Takaaki AU - Sato, Masahiro PY - 2007/12/28/ UR - http://dx.doi.org/10.1002/mrd.20847 ER -