Tetracycline-inducible system for regulation of skeletal muscle-specific gene expression in transgenic mice.
Tightly regulated control of over-expression is often necessary to study one aspect or time point of gene function and, in transgenesis, may help to avoid lethal effects and complications caused by ubiquitous over-expression. We have utilized the benefits of an optimized tet-on system and a modified muscle creatine kinase (MCK) promoter to generate a skeletal muscle-specific, doxycycline (Dox) controlled over-expression system in transgenic mice. A DNA construct was generated in which the codon optimized reverse tetracycline transactivator (rtTA) was placed under control of a skeletal muscle-specific version of the mouse MCK promoter. Transgenic mice containing this construct expressed rtTA almost exclusively in skeletal muscles. These mice were crossed to a second transgenic line containing a bi-directional promoter centered on a tet responder element driving both a luciferase reporter gene and a tagged gene of interest; in this case the calpain inhibitor calpastatin. Compound hemizygous mice showed high level, Dox dependent muscle-specific luciferase activity often exceeding 10,000-fold over non-muscle tissues of the same mouse. Western and immunocytochemical analysis demonstrated similar Dox dependent muscle-specific induction of the tagged calpastatin protein. These findings demonstrate the effectiveness and flexibility of the tet-on system to provide a tightly regulated over-expression system in adult skeletal muscle. The MCKrtTA transgenic lines can be combined with other transgenic responder lines for skeletal muscle-specific over-expression of any target gene of interest.
Grill
Mischala A
MA
Department of Cell Biology and Anatomy, PO Box 245044, University of Arizona, Tucson, Arizona, 85724, USA.
Bales
Mark A
MA
Fought
Amber N
AN
Rosburg
Kristopher C
KC
Munger
Stephanie J
SJ
Antin
Parker B
PB
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002154:Calpain / Q000037:antagonists & inhibitors; D002417:Cattle; D002478:Cells, Cultured; D003402:Creatine Kinase / Q000235:genetics* / Q000378:metabolism; D052283:Creatine Kinase, MM Form; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004318:Doxycycline / Q000494:pharmacology*; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects*; D017930:Genes, Reporter; D007124:Immunoenzyme Techniques; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D008822:Mice, Transgenic; D018482:Muscle, Skeletal / Q000201:enzymology*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*
D000900:Anti-Bacterial Agents; D002135:Calcium-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; D017931:DNA Primers; D007527:Isoenzymes; C033668:calpastatin; D008156:Luciferases; D003402:Creatine Kinase; D052283:Creatine Kinase, MM Form; D002154:Calpain; D004318:Doxycycline