Generation of both an shRNA-resistant MEF2A over expression construct and a dominant negative construct in adenovirus for rescue and knockout experiments in muscle
Comeau, Kathryn Marie
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The Myocyte Enhancer Factor-2, or MEF2, transcription factor family is necessary for the differentiation and regeneration of both skeletal and cardiac muscle tissue. The transcription factors in this family are responsible for the activation of many muscle specific growth factor-induced and differentiation genes. There are four individual isoforms of MEF2; MEF2A, -B, -C, and –D, and the roles of these individual transcription factors are not completely understood. Knockdowns of these individual isoforms revealed that a MEF2A knockdown mouse model displays severe myofibrillar defects in cardiac muscle. This knockdown also has shown that MEF2A is required for myogenesis in vitro, where the other 3 isoforms, -B, -C, and –D, are not necessary for this process. One method of knocking down MEF2A to study its roles further is through the use of short hairpin RNAs (shRNA). The purpose of my research was two-fold. First, in order to test the specificity of this shRNA method, an shRNA-resistant MEF2A over expression construct in an adenoviral vector was created to perform rescue experiments. Second, to compare individual MEF2 isoform knockouts to a complete knockout of the entire MEF2 family, a dominant negative construct was created in an adenoviral vector. In both cases, a pShuttle-CMV adenoviral vector was used. The results of this experiment can be used to further investigate the roles of MEF2A in both regeneration and differentiation of skeletal and cardiac muscle tissue.