Directing RNA interference specifically to differentiated muscle cells.
A common approach for mediating RNA interference (RNAi) is to introduce DNA that encodes short hairpin RNA (shRNA), which is often contained in a plasmid that can express a shRNA in a wide variety of cell types. Muscle cells and certain other cell types grown in culture can exist in both a dividing state and in a post-mitotic, differentiated state, and it is sometimes useful to induce RNAi selectively in terminally differentiated cells to study the function of a gene, particularly when the gene is also required for propagation of dividing cells. We describe two methods for studying gene function by RNAi specifically in terminally differentiated skeletal muscle cells in culture. We developed a shRNA expression vector, based on myosin light chain 1f gene regulatory sequences, which is designed to induce shRNA expression specifically after differentiation has been initiated. We show that this vector can mediate RNAi and is only active in differentiated muscle cells. Also, we developed an adenoviral vector that is designed to be able to deliver shRNAs directly to post-mitotic muscle cells. We show that adenoviruses produced using this vector mediate RNAi in differentiated muscle cells. These methods add to the repertoire of RNAi tools that can be used for identifying genes involved in any event of interest that occurs in differentiated muscle cells.
Herndon
Carter A
CA
Indiana University School of Medicine-Muncie, 2000 University Avenue, Muncie, IN 47306, USA.
Fromm
Larry
L
D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation; D002460:Cell Line; D020868:Gene Silencing; D018390:Gene Targeting / Q000379:methods*; D005821:Genetic Techniques; D005822:Genetic Vectors; D051379:Mice; D008969:Molecular Sequence Data; D032342:Muscle Cells / Q000166:cytology / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection
D034741:RNA, Small Interfering