Abstract
We have employed the hybrid hammerhead ribozyme-based gene discovery system for identification of genes functionally involved in muscle differentiation using in vitro myoblast differentiation assay. The major muscle regulatory genes (MyoD1, Mylk, myosin, myogenin, and Myf5) were identified endorsing the validity of this method. Other gene targets included tumor suppressors and cell cycle regulators (p19ARF and p21WAF1), FGFR-4, fibronectin, Prkg2, Pdk4, fem, and six novel proteins. Functional involvement of three of the identified targets in myoblast differentiation was confirmed by their specific knockdown using ribozymes and siRNA. Besides demonstrating a simple and an effective method of isolation of gene functions involved in muscle differentiation, we report for the first time that overexpression of Fem, a member of the sex-determining family of proteins, caused accelerated myotube formation, and its targeting deferred myoblast differentiation. This functional gene screening is not only helpful in understanding the molecular pathways of muscle differentiation but also to design molecular strategies for myopathologic therapies.
Highlights
Ribozymes (Rz)1 are small RNA molecules that catalyze the hydrolysis of specific phosphodiester bonds of RNA strands with which they form base pairing and act as specific molecular scissors providing a very useful tool of studying gene function in vitro and in vivo [1, 2]
Besides demonstrating a simple and an effective method of isolation of gene functions involved in muscle differentiation, we report for the first time that overexpression of Fem, a member of the sex-determining family of proteins, caused accelerated myotube formation, and its targeting deferred myoblast differentiation
The abbreviations used are: Rz, ribozyme; siRNA, small interfering RNA; pol, polymerase; ECM, extracellular matrix; FGF, fibroblast growth factor; retinoblastoma tumor suppressor protein (Rb), retinoblastoma; Hammerhead ribozymes (HH-Rz), hammerhead ribozymes. Of these 7–9 nucleotides in each arm yields a large variety of ribozymes capable of targeting multiple mRNA substrates
Summary
Construction of Randomized Hammerhead Ribozyme Libraries— Chemically synthesized oligonucleotides encoding ribozyme sequences with randomized substrate binding arms and a pol III termination sequence were converted to double-stranded sequences by PCR as described previously [27]. Cells were induced to undergo muscle differentiation by culturing in the medium supplemented with 2% horse serum. 2. Abrogation of C2C12 differentiation by randomized Rzs. C2C12 control (panel a) and randomized ribozyme library transduced (panel b) cells cultured in differentiation medium (panels c and d) are shown. Analysis of Functional Involvement of Genes during Muscle Differentiation—C2C12 cells were transfected with specific ribozyme or with siRNA gene knockdown constructs using LipofectAMINETM PLUS (Invitrogen). Transfected cells were selected in puromycin (2.5 g/ml)supplemented medium for 2– 4 days and subjected to differentiation. Expression of muscle-specific genes in control and gene knockdown cells were analyzed by Western blotting with specific antibodies as given below. Immunostaining—C2C12 cells and its derivatives (transfected with either expression or siRNA plasmids for fem as indicated) were induced for differentiation by incubating in the growth medium supplemented with 2% horse serum.
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