Myeloid cell promoter and constructs containing same

Abstract

The present invention provides novel myeloid cell specific promoters, and cis-acting elements that influence the activity of a myeloid cell specific promoters, as well as promoter-heterologous gene constructs and transfected myeloid cells which include the novel promoters and/or the novel cis-acting elements. The present invention also provides a method of transfecting myeloid cells and a method of producing a selected product within the transfected cells. The invention also includes a method for identifying factors that can regulate myeloid cell specific transcription.

1 . A promoter of the CD11d gene comprising all or a functional portion of isolated or recombinant SEQ ID NO:1, such that the sequence is sufficient to direct myeloid cell specific expression of a gene.  
     
     
         2 . The promoter of the CD11d gene according to  claim 1 , wherein the sequence comprises one or more modifications, such that the modified sequence retains sufficient activity to direct myeloid cell specific expression of a gene.  
     
     
         3 . A promoter of the CD11d gene according to  claim 1 , wherein the functional portion is selected from the group consisting of −946 to +74 of SEQ ID NO: 1, and −173 to +74 of SEQ ID NO: 1 and further wherein the sequence comprises one or more modifications, such that the modified sequence retains sufficient activity to direct myeloid cell specific expression of a gene.  
     
     
         4 . A promoter of the CD11d gene comprising an isolated or recombinant double-stranded DNA molecule wherein one of the strands hybridizes to all or a functional portion of SEQ ID NO:1.  
     
     
         5 . The promoter of the CD11d gene according to  claim 4 , wherein one of the strands of the DNA hybridizes to all or a functional portion of a sequence selected from the group consisting of 225 to 1244 of SEQ ID NO:1, and 998 to 1244 of SEQ ID NO:1.  
     
     
         6 . A cis-acting element that influences the activity of a myeloid cell specific promoter, comprising a functional portion of isolated or recombinant SEQ ID NO: 1, such that the element is sufficient to influence the activity of a myeloid cell specific promoter.  
     
     
         7 . The cis-acting element according to  claim 6 , wherein the cis-acting element comprises one or more modifications, such that the modified element retains sufficient activity to enhance the activity of a myeloid cell specific promoter.  
     
     
         8 . The cis-acting element according to  claim 6 , wherein the cis-acting element is a binding site for a transcription factor.  
     
     
         9 . The cis-acting element according to  claim 8 , wherein the cis-acting element is a binding site for Sp1.  
     
     
         10 . The cis-acting element according to  claim 6 , wherein the cis-acting element is a myeloid cell specific silencer element.  
     
     
         11 . A promoter of the CD11d gene comprising an isolated or recombinant double-stranded DNA molecule wherein one strand hybridizes to all or a functional portion of SEQ ID NO:1.  
     
     
         12 . A cell specific CD11d promoter-heterologous gene construct, the CD11d promoter comprising all or a functional portion of SEQ ID NO: 1 such that the sequence is sufficient to direct myeloid cell specific expression of a gene, and a heterologous gene, wherein expression of the heterologous gene of the construct is under transcriptional control of the CD11d promoter.  
     
     
         13 . The cell specific CD11d promoter-heterologous gene construct according to  claim 12 , wherein the functional portion of the CD11d promoter is selected from the group consisting of −946 to +74 of SEQ ID NO: 1, and −173 to +74 of SEQ ID NO:1.  
     
     
         14 . The cell specific CD11d promoter-heterologous gene construct according to  claim 12 , wherein the Cd11d promoter sequence comprises one or more modifications, such that the modified sequence retains sufficient activity to direct myeloid cell specific expression of a gene.  
     
     
         15 . The cell specific CD11d promoter-heterologous gene construct according to  claim 12 , wherein the construct further comprises a cis-acting element that influences the activity of the CD11d promoter, the cis-acting element located (5′) upstream of the heterologous gene.  
     
     
         16 . The cell specific CD11d promoter-heterologous gene construct according to  claim 12 , wherein the cis-acting element comprises a functional portion of isolated or recombinant SEQ ID NO:1, such that the element is sufficient to effect the activity of a myeloid cell specific promoter.  
     
     
         17 . The cell specific CD11d promoter-heterologous gene construct according to  claim 12 , wherein the cis-acting element is the −946 to +74 region of SEQ ID NO:1.  
     
     
         18 . The cell specific CD11d promoter-heterologous gene construct according to  claim 12 , wherein the cis-acting element is the −173 to +74 region of SEQ ID NO:1.  
     
     
         19 . The cell specific CD11d promoter-heterologous gene construct according to  claim 12 , wherein the cis-acting element is the −72 to −40 region of SEQ ID NO:1.  
     
     
         20 . The cell specific CD11d promoter-heterologous gene construct according to  claim 12 , wherein the cis-acting element comprises one or more modifications, such that the modified element retains sufficient activity to influence the activity of a myeloid cell specific promoter.  
     
     
         21 . The cell specific CD11d promoter-heterologous gene construct according to  claim 12 , wherein the cis-acting element is a binding site for a transcription factor.  
     
     
         22 . The cell specific CD11d promoter-heterologous gene construct according to  claim 21 , wherein the cis-acting element is a binding site for Sp1.  
     
     
         23 . The cell specific CD11d promoter-heterologous gene construct according to  claim 22 , wherein the binding site for Sp1 is the −63 to −40 (5′) region of SEQ ID NO:1.  
     
     
         24 . The cell specific CD11d promoter-heterologous gene construct according to  claim 12 , wherein the cis-acting element is a myeloid cell specific silencer element.  
     
     
         25 . The cell specific CD11d promoter-heterologous gene construct according to  claim 12 , wherein the cis-acting element is the −591 to −378 region of SEQ ID NO:1.  
     
     
         26 . A myeloid cell that expresses a heterologous gene product under transcriptional control of a CD11d promoter, wherein expression of the heterologous gene of the construct is under transcriptional control of the CD11d promoter.  
     
     
         27 . The myeloid cell according to  claim 26 , wherein the CD11d promoter comprises a double-stranded DNA molecule wherein one of strands has the sequence which hybridizes to all or a functional portion of the DNA sequence of SEQ ID NO:1.  
     
     
         28 . The myeloid cell according to  claim 26 , wherein the CD11d promoter is a functional portion of SEQ ID NO:1.  
     
     
         29 . The myeloid cell according to  claim 26 , wherein the functional portion is selected from the group consisting of −946 to +74 of SEQ ID NO:1, and −173 to +74 of SEQ ID NO:1.  
     
     
         30 . The myeloid cell according to  claim 26 , wherein the cell further comprises a cis-acting element that affects the CD11d promoter.  
     
     
         31 . The myeloid cell according to  claim 30 , wherein the cis-acting element is the −946 to +74 region of SEQ ID NO:1.  
     
     
         32 . The myeloid cell according to  claim 30 , wherein the cis-acting element is the −173 to +74 region of SEQ ID NO:1.  
     
     
         33 . The myeloid cell according to  claim 18 , wherein the cis-acting element is the −72 to −40 region of SEQ ID NO:1.  
     
     
         34 . A method of expression for myeloid cell specific genes, comprising: 
 (a) positioning a gene of interest so that it is functionally controlled by the promoter of  claim 1;  and    (b) expressing the gene of interest.    
     
     
         35 . A method of expression for myeloid cell specific genes, comprising: 
 (a) positioning a gene of interest so that it is functionally controlled by a promoter of  claim 1 , wherein the functional portion is selected from the group consisting of −946 to +74 of SEQ ID NO: 1, and −173 to +74 of SEQ ID NO: 1; and    (b) expressing the gene of interest.    
     
     
         36 . A method for identifying regulating factors of myeloid cell specific transcription, comprising: 
 (a) contacting a myeloid cell which contains a heterologous gene under the transcriptional control of a CD11d promoter with a selected factor, wherein the promoter comprises all or a functional portion of isolated or recombinant SEQ ID NO: 1, such that the sequence is sufficient to direct myeloid cell specific expression of the heterologous gene; and    (b) assaying for expression of the heterologous gene and comparing its expression in cells contacted with the factor with expression of the gene in cells not contacted with the factor, and thereby determining whether the expression pattern of the heterologous gene is altered in cells contacted with the factor as compared to that of cells which have not been contacted with the factor.    
     
     
         37 . A cis-acting element that influences the activity of a myeloid cell specific promoter, comprising a functional portion of isolated or recombinant SEQ ID NO: 2, such that the element is sufficient to influence the activity of a myeloid cell specific promoter.