Dopamine receptor type 2 specific promoter and methods of use thereof

Abstract

A nucleic acid containing a dopamine receptor type 2-specific promoter (D2SP) is provided. In certain embodiments, the nucleic acid includes a dopamine receptor type 2-specific promoter (D2SP), wherein the D2SP does not include exon 1 of a D2 receptor gene, wherein the D2SP comprises a Kozak sequence, and wherein the D2SP includes a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1. Also provided are expression vectors, genetically modified host cells and kits that include the subject nucleic acid.

What is claimed is: 
     
         1 . A nucleic acid comprising a dopamine receptor type 2-specific promoter (D2SP), wherein the D2SP does not include exon 1 of a D2 receptor gene, wherein the D2SP contains a Kozak sequence, and wherein the D2SP contains a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1. 
     
     
         2 . The nucleic acid of  claim 1 , wherein the Kozak sequence is at the 3′ terminus of the D2SP. 
     
     
         3 . The nucleic acid of any of  claims 1  and  2 , wherein the D2SP contains a BamHI restriction site. 
     
     
         4 . The nucleic acid of  claim 3 , wherein the BamHI restriction site is located 5′ of the Kozak sequence. 
     
     
         5 . The nucleic acid of any of  claims 1  to  4 , wherein the D2SP contains a nucleotide sequence having at least 98% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1. 
     
     
         6 . The nucleic acid of any of  claims 1  to  5 , wherein the D2SP is operably linked to a nucleotide sequence encoding a gene product that provides a detectable signal. 
     
     
         7 . The nucleic acid of  claim 6 , wherein the gene product that provides a detectable signal is a fluorescent protein. 
     
     
         8 . The nucleic acid of  claim 7 , wherein the fluorescent protein is selected from the group consisting of a green fluorescent protein, a yellow fluorescent protein, a cyan fluorescent protein, a calcium indicator and a voltage indicator. 
     
     
         9 . The nucleic acid of any of  claims 1  to  8 , wherein the D2SP is operably linked to a nucleotide sequence encoding a light-responsive polypeptide. 
     
     
         10 . The nucleic acid of  claim 9 , wherein the light-responsive polypeptide is a depolarizing light-responsive polypeptide, wherein the depolarizing light-responsive polypeptide contains an amino acid sequence having at least 75% sequence identity to any one of SEQ ID NOs: 4-37. 
     
     
         11 . The nucleic acid of  claim 9 , wherein the light-responsive polypeptide is a hyperpolarizing light-responsive polypeptide, wherein the hyperpolarizing light-responsive polypeptide contains an amino acid sequence having at least 75% sequence identity to any one of SEQ ID NOs: 38-56. 
     
     
         12 . The nucleic acid of any of  claims 1  to  11 , wherein the D2SP is operably linked to a nucleotide sequence encoding a recombinase. 
     
     
         13 . The nucleic acid of  claim 12 , wherein the recombinase is selected from the group consisting of a Cre recombinase and a FLP recombinase. 
     
     
         14 . A recombinant expression vector comprising the nucleic acid of any of  claims 1  to  13 . 
     
     
         15 . A genetically modified host cell comprising the nucleic acid of any of  claims 1  to  13 , or the recombinant expression vector of  claim 14 . 
     
     
         16 . The genetically modified host cell of  claim 15 , wherein the host cell is a neuronal cell. 
     
     
         17 . The genetically modified host cell of  claim 15 , wherein the host cell is a progenitor cell. 
     
     
         18 . The genetically modified host cell of  claim 17 , wherein the progenitor cell is a stem cell. 
     
     
         19 . A method of modulating activity of a target neuron, the method comprising introducing into the target neuron the nucleic acid of any of  claims 1  to  13 , wherein the D2SP is operably linked to a light-responsive polypeptide that, when activated by light, induces hyperpolarization or depolarization of the target neuron. 
     
     
         20 . A method of fluorescently labeling a target cell, the method comprising introducing into the target cell the nucleic acid of any of  claims 1  to  13 , wherein the D2SP is operably linked to a fluorescent protein that, when expressed, fluorescently labels the target cell. 
     
     
         21 . The method of  claim 20 , wherein the target cell is a neuronal cell. 
     
     
         22 . The method of  claim 20 , wherein the target cell is a progenitor cell. 
     
     
         23 . The method of  claim 22 , wherein the progenitor cell is a stem cell. 
     
     
         24 . A kit comprising:
 a recombinant expression vector that comprises a nucleic acid comprising a dopamine receptor type 2-specific promoter (D2SP), wherein the D2SP does not include exon 1 of a D2 receptor gene, wherein the D2SP contains a Kozak sequence, and wherein the D2SP comprises a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1; and   instructions for introducing the recombinant expression vector into a target cell.   
     
     
         25 . The kit of  claim 24 , wherein the kit further comprises a control expression vector that contains a nucleic acid containing a dopamine receptor type 2-specific promoter (D2SP), wherein the D2SP does not include exon 1 of a D2 receptor gene, wherein the D2SP comprises a Kozak sequence, and wherein the D2SP comprises a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1.