Myostatin regulatory region, nucleotide sequence determination and methods for its use

Abstract

The present invention relates to a transcription regulatory region of a myostatin gene. In particular, the invention relates to a 2.5 kb polynucleotide immediately 5′ to the myostatin coding sequence, its nucleotide sequence and methods of using this regulatory region, and fragments thereof. The present invention relates to the use of the myostatin promoter of the present invention to direct expression of a target gene in a tissue specific manner, i.e. muscle tissue. The present invention relates to the use of the myostatin promoter of the present invention in high throughput screens to identify test compounds which inhibit myostatin promoter activity or myostatin expression. In accordance with the present invention, inhibitors of the myostatin promoter may be used to inhibit myostatin expression as a method of engineering animals with increased lean meat in order to decrease the time required to bring the animals to slaughter, and of promoting muscle growth for treating disorders related to expression of the myostatin gene, such as muscle wasting associated with aging or disease in humans and companion animals, particularly cats and dogs.

1 . An isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 from 1 to 2482 (2.5 kb fragment) or fragment thereof that promotes transcription.  
     
     
         2 . An isolated polynucleotide which hybridizes under stringent conditions to SEQ ID NO. 1 and is capable of promoting transcription of a coding sequence when operatively linked to said coding sequence.  
     
     
         3 . An isolated polynucleotide which hybridizes under stringent conditions to SEQ ID NO. 3 and is capable of promoting transcription of a coding sequence in a muscle cell when operatively linked to said coding sequence, with the proviso that said polynucleotide is not derived from a porcine genome.  
     
     
         4 . A recombinant vector comprising the polynucleotide of  claim 1 ,  2  or 3.  
     
     
         5 . A recombinant expression vector comprising the polynucleotide of  claim 1 ,  2  or  3  in which the nucleotide sequence of the polynucleotide is operably associated with a coding sequence.  
     
     
         6 . A genetically engineered host cell comprising the vector of  claim 4  or  5 , or progeny thereof.  
     
     
         7 . The host cell of  claim 6  in which the coding sequence is a reporter gene.  
     
     
         8 . The host cell of  claim 7  in which the reporter gene is luciferase.  
     
     
         9 . A method of screening for an inhibitor of expression of a myostatin promoter region comprising: 
 (a) treating the host cell of  claim 7  with a test compound; and    (b) detecting a decrease of expression of a reporter gene under the control of the myostatin promoter region.    
     
     
         10 . A method of screening for an activator of expression of a myostatin promoter region comprising: 
 (a) treating the host cell of  claim 7  with a test compound; and    (b) detecting an increase of expression of a reporter gene under the control of the murine myostatin promoter region.    
     
     
         11 . The method of  claim 9  or  10  where the reporter gene is luciferase.  
     
     
         12 . The method of  claim 9  or  10  where the myostatin promoter is the murine myostatin promoter.  
     
     
         13 . A method of regulating myostatin gene expression comprising administering to a target cell a therapeutically effective amount of the inhibitor of a myostatin promoter region according to the method of  claim 9  or the activator of a myostatin promoter according to the method of  claim 10 .  
     
     
         14 . A method of inhibiting myostatin gene expression comprising introducing into a target cell a therapeutically effective amount of an agent that directly or indirectly inhibits the transcription activities of the polynucleotide of  claim 1 .  
     
     
         15 . A method of inhibiting myostatin gene expression comprising introducing into a target cell, via homologous recombination, a polynucleotide fragment homologous to the polynucleotide of  claim 1 , wherein the fragment inhibits the transcription activities of the polynucleotide of  claim 1 .  
     
     
         16 . A method of treatment of a neuromuscular disease in an organism comprising introducing into a target cell a therapeutically effective amount of an agent that inhibits the transcription activities or expression of the polynucleotide of  claim 1 .  
     
     
         17 . A method of treatment of cancer in an organism comprising introducing into a target cell a therapeutically effective amount of an agent that inhibits the transcription activities or expression of the polynucleotide of  claim 1 .  
     
     
         18 . A method of treatment of aging in an organism comprising introducing into a target cell a therapeutically effective amount of an agent that inhibits the transcription activities or expression of the polynucleotide of  claim 1 .  
     
     
         19 . A method of promoting muscle growth in an organism comprising introducing into a target cell an effective amount of an agent that inhibits the transcription activities or expression of the polynucleotide of  claim 1 .  
     
     
         20 . A method of promoting weight gain in an organism comprising introducing into a target cell an effective amount of an agent that inhibits the transcription activities or expression of the polynucleotide of  claim 1 .  
     
     
         21 . The method of  claim 15 ,  16 ,  17 ,  18 ,  19 , or  20  wherein the organism is selected from the group consisting of humans, dogs, cats, horses, cattle, sheep, pigs, chickens, fish, mice, rats, rabbits, guinea pigs, micro-pigs, goats, and non-human primates, including, baboons, monkeys and chimpanzees.  
     
     
         22 . A method for increasing the amount of lean meat in a food animal comprising administering to said food animal an effective amount of an agent that inhibits the transcription activities or expression of the polynucleotide of  claim 1 .  
     
     
         23 . The method of  claim 22 , wherein the food animal is selected from the group consisting of cattle, sheep, pigs, chickens, fish, and goats.  
     
     
         24 . A method of constructing a transgenic animal comprising introducing the expression vector of  claim 4  or  5  into an embryonic or other host cell.  
     
     
         25 . The transgenic animal of  claim 24 , wherein the expression of a transgene is regulated in a tissue specific manner.  
     
     
         26 . A method of expressing a heterologous gene in an animal in a tissue specific manner comprising introducing into said animal the expression vector of  claim 5 .  
     
     
         27 . A method of identifying compounds that modulate the activity of myostatin promoters and the expression of myostatin genes, wherein such method comprises administering a compound to a cell that expresses a gene under the control of a myostatin promoter or a transcriptionally active fragment thereof, measuring the level of gene expression or gene product activity and comparing this level to the level of gene expression or gene product activity produced by the cell in the absence of the compound, wherein if the level obtained in the presence of the compound differs from that obtained in its absence, a compound capable of modulating the expression of the myostatin gene or promoter activity is identified.  
     
     
         28 . The method of  claim 27  wherein the cell is the host cell of  claim 6.