Screening tools for discovery of novel anabolic agents

Abstract

The disclosure provides nucleic acids including the polynucleotide sequence of the human MAFbx core promoter involved in muscle specific expression. Also provided are reporters operably linked to a polynucleotide sequence including MAFbx transcription regulatory sequences, and constructs including polynucleotides that encode reporters and other polynucleotide sequences operably linked to the MAFbx core transcription regulatory sequence. Systems for identifying agents that inhibit muscle loss and/or increase muscle mass or tone are also provided.

1 . A method of identifying an agent, the method comprising:
 contacting a plurality of host cells with at least one test agent, wherein the plurality of host cells comprises a first recombinant nucleic acid molecule,
 wherein the first recombinant nucleic acid molecule comprises a hetrologous polynucleotide sequence encoding a first reporter, and wherein the first recombinant nucleic acid molecule is operably linked to a transcription regulatory sequence comprising the polynucleotide sequence of SEQ ID NO:1; and 
   detecting a decrease in expression of the first reporter as compared to a control, thereby identifying the agent as affecting skeletal muscle.   
   
   
       2 . The method of  claim 1 , wherein the transcription regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 2. 
   
   
       3 . The method of  claim 1 , wherein the first recombinant nucleic acid molecule comprises the polynucleotide sequence of SEQ ID NO:3. 
   
   
       4 . The method of  claim 1 , wherein the first reporter is luciferase, GFP, β-galactosidase, or β-glucuronidase. 
   
   
       5 . The method of  claim 1 , wherein the host cells are skeletal muscle cells. 
   
   
       6 . The method of  claim 1 , further comprising:
 identifying an agent that increases at least one of muscle mass and muscle tone by contacting with the test agent a plurality of host cells that comprise a second recombinant nucleic acid molecule,
 wherein the second recombinant nucleic acid molecule comprises a polynucleotide sequence encoding a second reporter operably linked to an Insulin-like growth factor I (IGF-1) transcription regulatory sequence; and 
   detecting an increase in expression of the second reporter operably linked to the IGF-1 transcription regulatory sequence as compared to a control, wherein the increase in expression indicates an agent that increases the at least one of muscle mass and muscle tone.   
   
   
       7 . The method of  claim 6 , wherein the plurality of host cells comprises at least a first host cell comprising the first recombinant nucleic acid molecule, and at least a second host cell comprising the second recombinant nucleic acid molecule. 
   
   
       8 . The method of  claim 7 , wherein the first and the second host cells are separately contacted with the same test agent. 
   
   
       9 . The method of  claim 7 , wherein both the first reporter and the second reporter are luciferase. 
   
   
       10 . The method of  claim 6 , wherein the plurality of host cells comprise both the first recombinant nucleic acid molecule and the second recombinant nucleic acid molecule. 
   
   
       11 . The method of  claim 10 , wherein the first reporter and the second reporter are different reporters selected from luciferase, GFP, β-galactosidase, and β-glucuronidase. 
   
   
       12 . A method for identifying an agent that affects the expression of a skeletal muscle gene, the method comprising:
 contacting a test agent with at least one isolated skeletal muscle cell or isolated skeletal muscle stem cell comprising a recombinant nucleic acid molecule,
 wherein the recombinant nucleic acid molecule comprises a hetrologous polynucleotide sequence encoding a reporter, and wherein the recombinant nucleic acid molecule is operably linked to a transcription regulatory sequence comprising the polynucleotide sequence of SEQ ID NO:1; and 
   detecting a decrease in expression of the reporter, thereby identifying an agent as affecting expression of a skeletal muscle gene.