Triple-fusion constructs and methods of monitoring human embryonic stem cells
Embodiments of the present disclosure include triple-fusion human embryonic stem cells, methods of imaging triple-fusion human embryonic stem cells, triple-fusion polynucleotides, triple-fusion proteins, methods of monitoring the progression of human embryonic stem cells, methods of making isolated triple-fusion human embryonic stem cells, and the like.
1. A method of monitoring human embryonic stem cells (hESCs) comprising:
providing human embryonic stem cells;
transducing the hESCs with a triple-fusion expression vector to produce triple-fusion hESCs, wherein the triple-fusion expression vector comprises a firefly luciferase reporter gene (Fluc) having a nucleotide sequence selected from the group consisting of: SEQ ID NO: 1 and SEQ ID NO: 3, a red fluorescent reporter gene (RFP) having a nucleotide sequence of SEQ ID NO: 6, and a truncated HSV1-tK positron emission tomography (PET) reporter gene (HSV1-tTK) having a nucleotide sequence selected from the group consisting of: SEQ ID NO: 7 and SEQ ID NO: 14; and
detecting fluorescence, bioluminescence, a nuclear signal from a PET reporter, or a combination thereof, emitted from the triple-fusion hESCs and any progeny of the triple-fusion hESCs.
2. The method of claim 1 , wherein the expression vector is LV-pUP-Fluc-RFP-tTK, where LV is lentivirus, pUP is constitutive human ubiquitin promoter, Fluc is firefly luciferase reporter gene, RFP is the red fluorescent reporter gene, and tTK is the truncated HSV1-tk PET reporter gene.
3. The method of claim 1 , wherein the triple-fusion expression vector comprises a gene having SEQ ID NO: 8.
4. The method of claim 1 , wherein the triple-fusion expression vector comprises a gene encoding a triple-fusion protein having SEQ ID NO: 9.
5. The method of claim 1 , wherein the triple-fusion expression vector comprises a gene encoding a triple-fusion protein having SEQ ID NO: 18.
6. A method of monitoring human embryonic stem cells (hESCs) in a subject comprising:
providing triple-fusion hESCs having a triple-fusion expression vector comprising a firefly luciferase reporter gene (Fluc) having a nucleotide sequence selected from the group consisting of: SEQ ID NO: 1 and SEQ ID NO: 3, a red fluorescent reporter gene (RFP) having a nucleotide sequence of SEQ ID NO: 6, and a truncated HSV1-tK positron emission tomography (PET) reporter gene (HSV1-tTK) having a nucleotide sequence selected from the group consisting of: SEQ ID NO: 7 and SEQ ID NO: 14;
administering the triple-fusion hESCs to the subject; and
detecting fluorescence, bioluminescence, a nuclear signal from a PET reporter, or a combination thereof, emitted from the triple-fusion hESCs and any progeny of the triple-fusion hESCs.
7. The method of claim 6 , wherein the expression vector is LV-pUP-Fluc-RFP-tTK, where LV is lentivirus, pUP is constitutive human ubiquitin promoter, Fluc is firefly luciferase reporter gene, RFP is the red fluorescent reporter gene, and tTK is the truncated HSV1-tk PET reporter gene.
8. The method of claim 6 , wherein the triple-fusion expression vector comprises a gene having SEQ ID NO: 8.
9. The method of claim 6 , wherein the triple-fusion expression vector comprises a gene encoding a triple-fusion protein having SEQ ID NO: 9.
10. The method of claim 6 , wherein the triple-fusion expression vector comprises a gene encoding a triple-fusion protein having SEQ ID NO: 18.
11. A triple-fusion construct comprising:
a firefly luciferase reporter gene (Fluc) having a nucleotide sequence selected from the group consisting of: SEQ ID NO: 1 and SEQ ID NO:3;
a red fluorescent reporter gene (RFP) having a nucleotide sequence SEQ ID NO: 6; and a truncated Herpes Simplex Virus 1 thymidine kinase positron emission tomography (PET) reporter gene (HSV1-tTK) having a nucleotide sequence selected from the group consisting of: SEQ ID NO: 7 and SEQ ID NO: 14.
12. A method of monitoring stem cells comprising:
providing stem cells;
transducing the stem cells with a triple-fusion expression vector to produce triple-fusion stem cells, wherein the triple-fusion expression vector comprises a firefly luciferase reporter gene (Fluc) having a nucleotide sequence selected from the group consisting of: SEQ ID NO: 1 and SEQ ID NO: 3, a red fluorescent reporter gene (RFP) having a nucleotide sequence of SEQ ID NO: 6, and a truncated HSV1-tK positron emission tomography (PET) reporter gene (HSV1-tTK) having a nucleotide sequence selected from the group consisting of: SEQ ID NO: 7 and SEQ ID NO: 14; and
detecting fluorescence, bioluminescence, a nuclear signal from a PET reporter, or a combination thereof, emitted from the triple-fusion stem cells and any progeny of the triple-fusion stem cells.
13. The method of claim 12 , wherein the stem cells are human stem cells.
14. A method of monitoring human stem cells in a subject comprising:
providing triple fusion human stem cells having a triple-fusion expression vector comprising a firefly luciferase reporter gene (Fluc) having a nucleotide sequence selected from the group consisting of: SEQ ID NO: 1 and SEQ ID NO: 3, a red fluorescent reporter gene (RFP) having a nucleotide sequence of SEQ ID NO: 6, and a truncated HSV1-tK positron emission tomography (PET) reporter gene (HSV1-tTK) having a nucleotide sequence selected from the group consisting of: SEQ ID NO: 7 and SEQ ID NO: 14;
administering the triple-fusion human stem cells to the subject; and
detecting fluorescence, bioluminescence, a nuclear signal from a PET reporter, or a combination thereof, emitted from the triple-fusion human stem cells and any progeny of the triple-fusion human stem cells.