Method and system for delivering nucleic acid into a target cell
Controlled delivery of nucleic acid into target cells is achieved by immobilizing the nucleic acid and the cells to a substrate. Improved control over delivery is achieved by immobilizing the nucleic acid to the substrate via complementary DNA binding interactions with an oligonucleotide linker.
1. A cell culture system comprising:
a substrate;
a nucleic acid molecule non-covalently attached to an oligonucleotide linker tethered to the substrate;
a target cell capable of taking up the nucleic acid molecule by endocytosis, the cell being adhered to the substrate via a cell adhesion linker; and
a culture medium compatible with survival of the target cell.
2. A culture system as recited in claim 1 , wherein the substrate is selected from the group consisting of a polymeric hydrogel and a self-assembled monolayer.
3. A culture system as recited in claim 1 , wherein the nucleic acid molecule is selected from the group consisting of a linear molecule and a circular molecule.
4. A culture system as recited in claim 1 , wherein the oligonucleotide linker comprises an inverted repeat.
5. A culture system as recited in claim 1 , wherein the target cell is a cultured stem cell.
6. A culture system as recited in claim 1 , wherein the target cell is a cultured human cell.
7. A culture system as recited in claim 1 , wherein the target cell is a cultured human stem cell.
8. A method for delivering nucleic acid into a target cell in a cell culture system, the system comprising a substrate, a nucleic acid molecule non-covalently attached to an oligonucleotide linker tethered to the substrate, a cell adhesion linker attached to the substrate and a target cell capable of taking up the nucleic acid molecule by endocytosis, the target cell being adhered to the substrate via the cell adhesion linker, and a culture medium compatible with target cell survival, the method comprising the step of:
adjusting culture conditions of the cell culture system, such that the target cell takes up the nucleic acid molecule.
9. A method as recited in claim 8 , wherein the substrate is selected from the group consisting of a polymeric hydrogel and a self-assembled monolayer.
10. A method as recited in claim 8 , wherein the nucleic acid molecule is selected from the group consisting of a linear molecule and a circular molecule.
11. A method as recited in claim 8 , wherein the oligonucleotide linker comprises an inverted repeat.
12. A method as recited in claim 8 , wherein the target cell is a cultured stem cell.
13. A method as recited in claim 8 , wherein the target cell is a cultured human cell.
14. A method as recited in claim 8 , wherein the target cell is a cultured human stem cell.
15. A method as recited in claim 8 , wherein the adjusting step includes the step of adjusting the culture temperature.
16. A method as recited in claim 8 , wherein the adjusting steps includes the step of adjusting the culture pH.
17. A method as recited in claim 8 , wherein the adjusting step includes the step of adding a competing nucleic acid molecule that competes with the attached nucleic acid molecule for complementary interaction with the oligonucleotide linker.
18. A method as recited in claim 8 , wherein the adjusting step includes the step of stimulating the cell culture system with focused ultrasound.
19. A method as recited in claim 8 , wherein the adjusting step includes the step of rendering labile a linkage between the linker and the substrate.
20. A culture system as recited in claim 1 , wherein the cell adhesion linker comprises a RGD peptide.
21. A culture system as recited in claim 20 , wherein the RGD peptide comprises SEQ ID NO: 1.
22. A method as recited in claim 8 , wherein the cell adhesion linker comprises a RGD peptide.
23. A method as recited in claim 22 , wherein the RGD peptide comprises SEQ ID NO: 1.