Upstream control elements of the proopiomelanocortin gene and their use
View Patent ↗The nucleic acid sequence of the POMC enhancer is disclosed herein. Sequences from the human, rat, rabbit, hamster, mouse, and cow POMC enhancer are disclosed. Hybrid transgenes, comprising a POMC transcriptional control element operably linked to a nucleic acid sequence encoding a marker are also enclosed. In addition, transgenic mice carrying a hybrid transgene including a POMC control element operably linked to a marker are disclosed herein.
1. An isolated proopiomelanocortin enhancer element, comprising the nucleic acid sequence set forth as SEQ ID NO: 9, wherein the isolated proopiomelanocortin enhancer element elevates transcription of a heterologous coding sequence in a host cell, wherein the proopiomelanocortin element is isolated from the adjacent 5′ or 3′ nucleic acid sequences of an endogenous chromosome comprising the proopiomelancortin gene.
2. The isolated proopiomelanocortin enhancer element of claim 1 , consisting of the nucleic acid sequence set forth as SEQ ID NO: 9.
3. The isolated proopiomelanocortin enhancer element of claim 1 , comprising a 5′ half and a 3′ half, wherein the 5′ half and the 3′ half have the nucleic acid sequence set forth, respectively, as
a) SEQ ID NO: 1 and SEQ ID NO: 6;
b) SEQ ID NO: 4 and SEQ ID NO: 7; or
c) SEQ ID NO: 5 and SEQ ID NO: 8.
4. The isolated proopiomelanocortin enhancer element of claim 1 , comprising nucleic acid sequence as set forth as SEQ ID NO: 1 and the nucleic acid sequence set forth as SEQ ID NO: 6.
5. The isolated proopiomelanocortin enhancer element of claim 1 , further comprising
a) an nPOMC2 element comprising the nucleic acid sequence as set forth as one of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, or
b) an nPOMC3 element comprising the nucleic acid sequence as set forth as one of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19; or
c) a combination thereof
wherein the enhancer element directs expression of a heterologous nucleic acid sequence in a proopiomelanocortin neuron.
6. The isolated enhancer element of claim 1 , comprising a nucleic acid sequence as set forth as SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22.
7. The isolated enhancer element of claim 1 , operably linked to a promoter and a heterologous nucleic acid sequence encoding a polypeptide, wherein the enhancer element directs expression of the heterologous nucleic acid sequence encoding the polypeptide in a proopiomelanocortin neuron.
8. The isolated enhancer element of claim 7 , wherein the polypeptide is an enzyme, a marker, a polypeptide that confers antibiotic resistance, or an antigen.
9. The isolated enhancer element of claim 7 , wherein the polypeptide is green fluorescent protein.
10. The isolated enhancer element of claim 1 , operably linked to a heterologous promoter.
11. An expression vector comprising the isolated enhancer element of claim 1 operably linked to a heterologous promoter.
12. The vector of claim 11 , wherein the vector is a viral vector.
13. The vector of claim 11 , wherein the vector is a plasmid vector.
14. An isolated host cell transformed with the vector of claim 11 .
15. The isolated host cell of claim 14 , wherein the host cell is a eukaryotic cell.
16. The isolated host cell of claim 14 , wherein the host cell is a prokaryotic cell.
17. An expression cassette comprising the POMC enhancer of claim 1 , and a nucleic acid sequence encoding proopiomelanocortin comprising at least one exon, wherein a polylinker comprising a unique site for a restriction enzyme is inserted into the exon.
18. An isolated proopiomelanocortin enhancer element, comprising the nucleic acid sequence set forth as SEQ ID NO: 1, wherein the isolated proopiomelanocortin enhancer element elevates transcription of a heterologous coding sequence in a host cell, wherein the propiomelanocortin element is isolated from the adjacent 5′ or 3′ nucleic acid sequences of an endogenous chromosome comprising the proopiomelanocortin gene.
19. The isolated enhancer element of claim 18 , operably linked to a promoter and a heterologous nucleic acid sequence encoding a polypeptide, wherein the enhancer element directs expression of the heterologous nucleic acid sequence encoding the polypeptide in a proopiomelanocortin neuron.
20. The isolated enhancer element of claim 19 , wherein polypeptide is an enzyme, a marker, a polypeptide that confers antibiotic resistance, or an antigen.
21. The isolated enhancer element of claim 19 , wherein the polypeptide is green fluorescent protein.
22. The isolated enhancer element of claim 19 , operably linked to a heterologous promoter.
23. An expression vector comprising the isolated enhancer element of claim 19 operably linked to a heterologous promoter.
24. The vector of claim 23 , wherein the vector is a viral vector.
25. The vector of claim 23 , wherein the vector is a plasmid vector.
26. An isolated host cell transformed with the vector of claim 23 .
27. The isolated host cell of claim 26 , wherein the host cell is a eukaryotic cell.
28. The isolated host cell of claim 27 , wherein the host cell is a prokaryotic cell.