Multimodality imaging of reporter gene expression using a novel fusion vector in living cells and animals
Novel double and triple fusion reporter gene constructs harboring distinct imagable reporter genes are provided, as well as applications for the use of such double and triple fusion constructs in living cells and in living animals using distinct imaging technologies.
1. A double fusion nucleic acid expression vector, comprising:
a first reporter nucleic acid sequence, a second reporter nucleic acid sequence, and a linker sequence, wherein:
(i) the first reporter nucleic acid sequence, the second reporter nucleic acid sequence, and the nucleic acid linker sequence, are operably linked to an expression control system and co-expressible as a single fusion polypeptide therefrom;
(ii) the first reporter nucleic acid sequence and the second reporter nucleic acid sequence are separated from each other by the nucleic acid linker sequence;
(iii) each of the first and second reporter nucleic acid sequences encodes an individual imageable reporter region of the expressed fusion polypeptide;
(iv) each of the individual imageable reporter regions of the fusion polypeptide is imageable by a method selected from the group consisting of: detecting bioluminescence, detecting fluorescence, and positron emission topography, wherein no two reporter regions are imageable by the same method; and
(v) the nucleic acid linker sequence encodes a polypeptide linker having an amino acid sequence selected from the group consisting of: SEQ ID NOs: 9 and 10.
2. The double fusion expression vector of claim 1 , wherein the nucleic acid sequence encoding the imageable reporter region of the fusion polypeptide that is imageable by positron emission topography encodes herpes simplex virus type 1 sr39 thymidine kinase (HSVI-sr39 thymidine kinase).
3. The double fusion expression vector of claim 1 , wherein the first nucleic acid encodes wild type HSVI thymidine kinase.
4. The double fusion expression vector of claim 1 , wherein the first nucleic acid encodes a bioluminescent polypeptide.
5. The double fusion expression vector of claim 4 , wherein the first nucleic acid encodes renilla luciferase.
6. The double fusion expression vector of claim 4 , wherein the first nucleic acid encodes firefly luciferase.
7. The double fusion expression vector of claim 1 , wherein the first nucleic acid encodes a fluorescence polypeptide.
8. The double fusion expression vector of claim 7 , wherein the first nucleic acid encodes a red fluorescence protein.
9. The double fusion expression vector of claim 7 , wherein the first nucleic acid encodes a green fluorescence protein.
10. A cultured cell comprising the vector of claim 1 .
11. A cultured cell transfected with the vector of claim 1 , or a progeny of said cell, wherein the cell expresses the double fusion polypeptide.
12. An isolated non-human cell comprising the vector of claim 1 .
13. An isolated non-human cell transfected with the vector of claim 1 , or a progeny of said cell, wherein the cell expresses the double fusion polypeptide.
14. A double fusion expression vector comprising:
a first reporter nucleic acid sequence, wherein the first reporter nucleic acid sequence encodes a synthetic renilla luciferase;
a second reporter nucleic acid sequence, wherein the second reporter nucleic acid sequence encodes a red fluorescence protein;
a nucleic acid linker sequence, wherein:
i) the first reporter nucleic acid sequence, the second reporter nucleic acid sequence, and the nucleic acid linker sequence are operably linked to an expression control system and co-expressible as a single fusion polypeptide therefrom, and wherein the order of transcription from the expression control system is the first nucleic acid sequence and the second nucleic acid sequence;
(ii) the first reporter nucleic acid sequence and the second reporter nucleic acid sequence are separated from each other by the nucleic acid linker sequence; and
(iii) the nucleic acid linker sequence encodes a polypeptide linker having an amino acid sequence selected from the group consisting of: SEQ ID NOs: 9 and 10.