title: Analysis of Nuclear Lamina Proteins in Myoblast Differentiation by Functional Complementation.
abstract: We describe straightforward methodology for structure-function mapping of nuclear lamina proteins in myoblast differentiation, using populations of C2C12 myoblasts in which the endogenous lamina components are replaced with ectopically expressed mutant versions of the proteins. The procedure involves bulk isolation of C2C12 cell populations expressing the ectopic proteins by lentiviral transduction, followed by depletion of the endogenous proteins using siRNA, and incubation of cells under myoblast differentiation conditions. Similar methodology may be applied to mouse embryo fibroblasts or to other cell types as well, for the identification and characterization of sequences of lamina proteins involved in functions that can be measured biochemically or cytologically.
authors.affiliation: Department of Cell and Molecular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd, La Jolla, CA, 92037, USA.
authors.forename: Olga
authors.initials: O
authors.lastname: Tapia
authors.affiliation: Department of Cell and Molecular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd, La Jolla, CA, 92037, USA. lgerace@scripps.edu.
authors.forename: Larry
authors.initials: L
authors.lastname: Gerace
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D018482:Muscle, Skeletal / Q000378:metabolism; D032446:Myoblasts / Q000166:cytology* / Q000378:metabolism*; D034881:Nuclear Lamina / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D014161:Transduction, Genetic; D014162:Transfection
chemical_list: D008565:Membrane Proteins; D009687:Nuclear Proteins; D034741:RNA, Small Interfering
keywords: Functional complementation; Lentivirus; Myoblast; Nuclear lamina; siRNA