title: Forced MyHCIIB expression following targeted genetic manipulation of conditionally immortalized muscle precursor cells.
abstract: The ability to carry out gene targeting in somatic stem cells while maintaining their stem cell characteristics would have important implications for gene therapy and for the analysis of gene function. Using mouse myoblasts, we have explored this possibility by attempting to alter the promoter of a myosin heavy chain gene (MyHCIIB) characteristic of physiologically "fast" muscle so as to force its unscheduled expression in physiologically "slow" muscle fibers. Conditionally immortalized muscle precursor cells were transfected with a gene targeting construct designed to replace the MyHCIIB promoter with that for the carbonic anhydrase III gene (CAIII), which is highly expressed in slow muscle. A potentially targeted clone was isolated and differentiated in culture to form myotubes which expressed MyHCIIB. Cells from the same clone were injected into both slow and fast muscle of host mice, where they contributed to fiber formation. In slow muscle, the fibers derived from this clone did not express MyHCIIB; this may reflect an instability of the targeted MyHCIIB locus and/or a failure of the hybrid promoter to function in slow fibers in vivo. Nonetheless, we have demonstrated that a "promoter knock-in" gene targeting procedure can be used to generate unique MyHCIIB-expressing myotubes in culture and that conditionally immortalized myoblasts can be subjected to extensive passaging and genetic manipulation without losing their ability to form fibers in culture and in vivo.
authors.affiliation: MRC Clinical Sciences Centre, Hammersmith Hospital, London, W12 ONN, United Kingdom.
authors.forename: J M
authors.initials: JM
authors.lastname: Harris
authors.forename: J E
authors.initials: JE
authors.lastname: Morgan
authors.forename: J D
authors.initials: JD
authors.lastname: Rosenblatt
authors.forename: M
authors.initials: M
authors.lastname: Peckham
authors.forename: Y H
authors.initials: YH
authors.lastname: Edwards
authors.forename: T A
authors.initials: TA
authors.lastname: Partridge
authors.forename: A C
authors.initials: AC
authors.lastname: Porter
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D002461:Cell Line, Transformed / Q000737:chemistry / Q000166:cytology; D003001:Cloning, Molecular / Q000379:methods; D015870:Gene Expression / Q000502:physiology; D018014:Gene Transfer Techniques; D051379:Mice; D018656:Muscle Fibers, Fast-Twitch / Q000737:chemistry / Q000166:cytology*; D018657:Muscle Fibers, Slow-Twitch / Q000737:chemistry / Q000166:cytology*; D018482:Muscle, Skeletal / Q000166:cytology; D016254:Mutagenesis, Insertional / Q000502:physiology; D018995:Myosin Heavy Chains / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000502:physiology; D013234:Stem Cells / Q000737:chemistry / Q000166:cytology*; D014162:Transfection
chemical_list: D018995:Myosin Heavy Chains