title: A versatile single-plasmid system for tissue-specific and inducible control of gene expression in transgenic mice.
abstract: We describe a novel transgenic system for tissue-specific and inducible control of gene expression in mice. The system employs a tetracycline-responsive CMV promoter that controls transcription of a short-hairpin RNA (shRNA) that remains nonfunctional until an interrupting reporter cassette is excised by Cre recombinase. Insertion of Dicer and Drosha RNase processing sites within the shRNA allows generation of siRNA to knock down a target gene efficiently. Tissue-specific shRNA expression is achieved through the use of appropriate inducer mice with tissue-specific expression of Cre. We applied this system to regulate expression of junctophilins (JPs), genes essential for maintenance of membrane ultrastructure and Ca(2+) signaling in muscle. Transgenic mice with skeletal muscle-specific expression of shRNA against JP mRNAs displayed no basal change of JP expression before treatment with doxycycline (Dox), while inducible and reversible knockdown of JPs was achieved by feeding mice with Dox-containing water. Dox-induced knockdown of JPs led to abnormal junctional membrane structure and Ca(2+) signaling in adult muscle fibers, consistent with essential roles of JPs in muscle development and function. This transgenic approach can be applied for inducible and reversible gene knockdown or gene overexpression in many different tissues, thus providing a versatile system for elucidating the physiological gene function in viable animal models.
authors.affiliation: Department of Physiology and Biophysics, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.
authors.forename: Jae-Kyun
authors.initials: JK
authors.lastname: Ko
authors.forename: Kyoung-Han
authors.initials: KH
authors.lastname: Choi
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Zhao
authors.forename: Shinji
authors.initials: S
authors.lastname: Komazaki
authors.forename: Zui
authors.initials: Z
authors.lastname: Pan
authors.forename: Noah
authors.initials: N
authors.lastname: Weisleder
authors.forename: Jianjie
authors.initials: J
authors.lastname: Ma
mesh_terms: D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D017930:Genes, Reporter; D005821:Genetic Techniques; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic / Q000235:genetics*; D046529:Microscopy, Electron, Transmission; D018482:Muscle, Skeletal / Q000378:metabolism / Q000648:ultrastructure; D010957:Plasmids / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D014018:Tissue Distribution
chemical_list: D008565:Membrane Proteins; D034741:RNA, Small Interfering; C412507:junctophilin