Cre-loxP system as a versatile tool for conferring increased levels of tissue-specific gene expression from a weak promoter.
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.
Nakamura
Shingo
S
Department of Surgery, School of Medicine, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama, Japan.
Watanabe
Satoshi
S
Ohtsuka
Masato
M
Maehara
Tadaaki
T
Ishihara
Masayuki
M
Yokomine
Takaaki
T
Sato
Masahiro
M
D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D018274:Electroporation; D005260:Female; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D019426:Integrases; D008156:Luciferases / Q000235:genetics; D051379:Mice; D041681:NIH 3T3 Cells; D010057:Oviducts / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011817:Rabbits; D011995:Recombination, Genetic; D014018:Tissue Distribution; D014162:Transfection; D019076:Transgenes
D018076:DNA, Complementary; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D008156:Luciferases; C045073:Cre recombinase; D019426:Integrases