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P53/MyoD Functional Redundancy in Myogenesis

Mark Lopez and Sandra B. Sharp

The differentiation process of skeletal myogenesis is well known to be governed by both tissue specific and generally expressed regulatory factors.  In vivo and in vitro, cells are determined to become muscle by expression of MyoD and/or myf-5, two of the four members of the tissue specific myogenesis regulatory factor (MRF) family that also includes myogenin and MRF4.  Experiments with both primary and immortal myoblasts have strongly suggested that p53, which is generally expressed, is an important contributor to the success of myogenesis.  However, in apparent contrast, whole mouse p53 gene knockout experiments have revealed no obvious role for p53 in myogenesis.  Because p53 and MyoD are known to be redundant with respect to some activities, we hypothesize that expression of either p53 or MyoD is required for successful completion of myogenesis during development in vivo.  If our hypothesis is correct, then failure to express both MyoD and p53 would be expected to result in abnormal myogenesis in vivo, as evidenced by reduced levels of mRNA for other muscle specific regulatory factors or structural proteins that are upregulated during myogenesis and are necessary for normal muscle development to occur.  We are breeding p53/MyoD double knockout mice.  As a first test of our hypothesis, we will assess muscle development in newborn double knockouts by determining levels of accumulation of mRNAs for the four MRFs and other skeletal muscle specific proteins whose expression is required for normal myogenesis.  After euthanasia, total RNA from forelimbs, hindlimbs, tongue, back, and heart (control) will be isolated from both wild type and double knockout mice.  Accumulation of these skeletal muscle specific mRNAs will be determined using quantitative reverse transcriptase PCR.  Levels of PCR products will be quantified using BioRad iQ thermocycler real time detection of SYBER Green fluorescence and normalized to the expression levels of the housekeeping genes GAPDH and ribosomal protein L7.  The normalized levels of the muscle-specific mRNAs will be compared between p53/MyoD null and wild type mice.  The assay for each mRNA being investigated needs to be developed.  Thus far we have designed PCR primers for each of the mRNAs under investigation, picking them to flank introns.  The use of primers that flank introns controls for contributions from contaminating genomic DNA template.  We have determined optimal PCR conditions for several of the primer sets.  Each assay needs to be sufficiently sensitive to detect very small amounts of PCR products in order to ensure accurate comparison of RNA levels.  We will therefore determine the sensitivity for each set of primers using dilutions of mRNA extracted from newborn wild type mice. To be prepared to work on tissue from the double knockouts by the time they have been bred, I am currently optimizing all the primer sets with respect to annealing temperatures of the PCR primers, Mg++ concentrations for specificity of PCR, and sensitivity for low concentrations of mRNA.