Methods of screening for introduction of DNA into a target cell
View Patent ↗The present invention provides methods of introducing a polynucleotide into a target cell, wherein the method employs a light generating protein coding sequence acting as a reporter. An important advantage of the methods described herein is that drug resistant target cells or target cells having no useful auxotrophic markers can be effectively transformed. The present invention also includes transformed cells produced by the methods described herein. Also described are light generating protein coding sequence modifications, a variety of vectors, and methods of using the transformed cells of the present invention.
1. A method of isolating cells, said method comprising
providing a population of live yeast cells;
treating said population of live yeast cells with a polynucleotide under conditions which facilitate uptake of the polynucleotide by at least a sub-population of the cells, wherein said polynucleotide comprises a promoterless polynucleotide sequence encoding a luciferase protein, and further wherein only cells that take up the polynucleotide express light;
measuring photon emission from live yeast cells that have taken up the polynucleotide; and
isolating cells that produce light.
2. The method of claim 1 , wherein said yeast is Candida albicans.
3. The method of claim 1 , wherein said polynucleotide comprises an integrating vector.
4. The method of claim 1 , wherein said polynucleotide comprises a transposable or mobile genetic element, wherein said transposable or genetic element comprises said luciferase protein coding sequence.
5. A method of isolating cells, said method comprising
providing a population of live yeast cells;
treating said population of live yeast cells with a polynucleotide under conditions which facilitate uptake of the polynucleotide by at least a sub-population of the cells, wherein said polynucleotide comprises an integrating vector, the integrating vector comprising a promoter operably linked to a sequence encoding a luciferase protein, and further wherein only cells that take up the polynucleotide express light;
measuring photon emission from live yeast cells that have taken up the polynucleotide; and
isolating cells that produce light.
6. A method of isolating cells, said method comprising
providing a population of live yeast cells;
treating said population of live yeast cells with a polynucleotide under conditions which facilitate uptake of the polynucleotide by at least a sub-population of the cells, wherein said polynucleotide comprises a transposable or mobile genetic element, wherein said transposable or genetic element comprises said promoter operably linked to a luciferase protein coding sequence, and further wherein only cells that take up the polynucleotide express light;
measuring photon emission from live yeast cells that have taken up the polynucleotide; and
isolating cells that produce light.
7. A method of isolating cells, said method comprising
providing a population of live yeast cells;
treating said population of live yeast cells with a polynucleotide under conditions which facilitate uptake of the polynucleotide by at least a sub-population of the cells, the polynucleotide comprising a promoter operably linked to a sequence encoding a luciferase protein, wherein said polynucleotide comprises a circular plasmid having an origin of replication, and further wherein only cells that take up the polynucleotide express light;
measuring photon emission from live cells that have taken up the polynucleotide; and
isolating cells that produce light.
8. The method of claim 7 , wherein said origin of replication is functional in said cells.
9. The method of claim 1 , wherein said polynucleotide further comprises a first coding sequence of interest.
10. The method of claim 9 , wherein said first coding sequence of interest is operably linked to a promoter functional in said cells.
11. The method of claim 1 , and further wherein photon emission is measured when the live yeast cells are on a plate of solid medium and said isolating comprises picking samples of cells from a region of the plate of solid medium comprising cells producing light, inoculating said samples into liquid cultures, growing the cells in liquid cultures, evaluating aliquots of each liquid culture for light production, identifing liquid cultures comprising cells producing light, diluting and plating liquid cultures to obtain isolated single cells producing light, and identifying single colonies derived from single cells producing light.
12. A method of screening for transformed cells in a population of cells, said method comprising
providing a population of live yeast cells, wherein said cells are incapable of producing light;
treating said population of live yeast cells with a polynucleotide under conditions that facilitate uptake of the polynucleotide by at least a sub-population the cells, wherein said polynucleotide comprises a luciferase coding sequence and a heterologous gene of interest; and
screening said treated population of live yeast cells for cells that produce light from said luciferase coding sequence, wherein said live cells that produce light are transformed cells.
13. The method of claim 12 , wherein the yeast cells are Candida albicans cells.