Development of a ponasterone A-inducible gene expression system for application in cultured skeletal muscle cells.
The goal of this study was to develop an inducible gene expression system to assess functions of specific proteins in differentiated cultured skeletal muscle. We utilized and modified the ecdysone inducible system because others have used this system to express exogenous genes in vitro and in transgenic animals. A limitation of the commercially-available ecdysone system is its constitutive expression in all tissues. Hence, its application in vivo would result in expression of a cloned gene in undifferentiated and differentiated tissues. To target its expression to muscle, we removed the constitutively-active CMV promoter of pVgRXR and replaced it with a skeletal muscle alpha-actin promoter so that the regulatory features of the system would be expressed in differentiated muscle cells. We transfected our newly designed expression system into L8 muscle myoblasts and established stable cell lines via antibiotic selection. We determined that reporter gene activity was induced by ponasterone A in myotubes, a differentiated muscle phenotype, but not in myoblasts (undifferentiated cells). This proved the validity of the concept of an inducible muscle-specific expression system. We then determined that beta-galactosidase expression was dependent upon the dose of ponasterone A and duration of exposure to inducer. This creates potential to regulate both the level of expression and duration of expression of a cloned gene in differentiated muscle.
Xiao
Ying-yi
YY
Department of Animal Sciences, Oregon State University, Corvallis, OR 97331-6702, USA. xiaoy10@hotmail.com
Beilstein
Michael A
MA
Wang
Mei-chuan
MC
Purintrapiban
Juntipa
J
Forsberg
Neil E
NE
D000199:Actins / Q000187:drug effects / Q000235:genetics; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004441:Ecdysterone / Q000031:analogs & derivatives / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects*; D005818:Genetic Engineering / Q000379:methods; D018482:Muscle, Skeletal / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D032446:Myoblasts / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D051381:Rats; D014162:Transfection; D001616:beta-Galactosidase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism
D000199:Actins; D004441:Ecdysterone; C008999:ponasterone A; D001616:beta-Galactosidase