Methods of screening for introduction of DNA into a target cell

• US 20020192755A1
• Filed: 03/07/2002
• Published: 12/19/2002
• Est. Priority Date: 03/07/2001
• Status: Active Grant

• Current Assignee: Xenogen Corporation (Revvity, Inc.)
• Original Assignee: Xenogen Corporation (Revvity, Inc.)
• Inventors: Doyle, Timothy C., Nawotka, Kevin A., Francis, Kevin P.
• Granted Patent: US 7,090,994 B2
• Time in Patent Office: Days
• US Class Current: 435/69.1
• CPC Class Codes: C12N 15/74 Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
• CPC Class Codes: C12N 15/815 for yeasts other than Saccharomyces
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: G01N 2333/90241 acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
• CPC Class Codes: G01N 2500/10 involving cells

Abstract

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.

Claims

1. A method of introducing a polynucleotide into a cell, said method comprising providing a population of cells;

treating said population of 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 light generating protein coding sequence;

screening said population of cells for cells that produce light; and

isolating cells that produce light, wherein the cells capable of producing light are cells into which the polynucleotide has been introduced.

2. The method of claim 1, wherein said light generating protein is a bioluminescent or fluorescent protein.

3. The method of claim 2, wherein said bioluminescent protein is a luciferase.

4. The method of claim 1, wherein said population of cells is a population of procaryotic cells.

5. The method of claim 4, wherein said procaryotic cells are antibiotic-resistant bacteria.

6. The method of claim 4, wherein said procaryotic cells are gram-negative or gram-positive bacteria.

7. The method of claim 6, wherein said procaryotic cells are gram-positive bacteria.

8. The method of claim 7, wherein said gram-positive bacteria are of the species Staphylococcus.

9. The method of claim 1, wherein said population of cells is a population of eucaryotic cells.

10. The method of claim 9, wherein said population of cells is a yeast.

11. The method of claim 10, wherein said yeast is Candida albicans.

12. The method of claim 9, wherein said eucaryotic cells are mammalian cells.

13. The method of claim 1, wherein said polynucleotide comprises a promoterless polynucleotide sequence encoding a light generating protein.

14. The method of claim 13, wherein said polynucleotide comprises an integrating vector.

15. The method of claim 13, wherein said polynucleotide comprises a transposable or mobile genetic element, wherein said transposable or genetic element comprises said light generating protein coding sequence.

16. The method of claim 1, wherein said polynucleotide comprises a promoter operably linked to said sequences encoding a light generating protein.

17. The method of claim 16, wherein said polynucleotide comprises an integrating vector.

18. The method of claim 16, wherein said polynucleotide comprises a transposable or mobile genetic element, wherein said transposable or genetic element comprises said promoter operably linked to said light generating protein coding sequence.

19. The method of claim 16, wherein said polynucleotide comprises a circular plasmid.

20. The method of claim 19, wherein said plasmid further comprises an origin of replication functional in said cells.

21. The method of claim 1, wherein said polynucleotide further comprises a first coding sequence of interest.

22. The method of claim 21, wherein said first coding sequence of interest is operably linked to a promoter functional in said cells.

23. The method of claim 1, wherein said screening takes place 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, identifying 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.

24. A method of screening for transformed cells in a population of cells, said method comprising providing a population of cells, wherein said cells are incapable of producing light;

treating said population of 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 light generating protein coding sequence; and

screening said treated population of cells for cells producing light, wherein said cells that produce light are transformed cells.