METHOD FOR CONCENTRATING CELLS THAT ARE GENETICALLY ALTERED BY NUCLEASES

• US 20140045176A1
• Filed: 02/22/2012
• Published: 02/13/2014
• Est. Priority Date: 02/22/2011
• Status: Active Grant

• Current Assignee: Toolgen Incorporated
• Original Assignee: Toolgen Incorporated
• Inventors: Kim, Jin Soo, Kim, Seok Joon, Kim, Yong Sub
• Granted Patent: US 9,809,839 B2
• Time in Patent Office: Days
• US Class Current: 435/6.1
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• CPC Class Codes: C12Q 1/44 involving esterase
• CPC Class Codes: G01N 33/50 Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing measuring or testing processes involving enzymes or microorganisms, compositions or test papers therefor; processes for forming such compositions, condition responsive control in microbiological or enzymological processes C12Q

Abstract

The present invention relates to a reporter construct and method for identifying or enriching the cells, wherein a specific endogenous nucleotide sequence is cleaved by a specific nuclease or modified by such cleavage; a host cell comprising the reporter construct; and a system for monitoring a nuclease activity. The reporter system of the present invention is simple and non-invasive, and allows for an efficient enrichment of the gene-modified cells. Therefore, the present invention will promote the application of a nuclease in the field of gene therapy and genetic engineering as well as basic research.

Claims

1. A method for identifying or enriching the cells, wherein a specific endogenous nucleotide sequence is cleaved by a specific nuclease or modified by such cleavage, comprising

a first step of preparing a reporter construct comprising a target sequence recognized by the nuclease and a reporter gene, wherein the expression of the reporter gene in the reporter construct is determined by the binding of the nuclease to the target sequence and cleaving the reporter construct;

a second step of inserting the reporter construct into a candidate cell, wherein a portion or all of the candidate cells express the nuclease before or after inserting the reporter construct; and

a third step of sorting the candidate cells obtained from step 2 into the cells expressing the reporter gene or the cells not expressing the reporter gene.

2. The method according to claim 1, wherein the specific endogenous nucleotide sequence is an intrinsic nucleotide sequence present in the genome.

3. The method according to claim 1, wherein the reporter construct has the target sequence, which is recognized by the nuclease, inserted in the middle or in front of the reporter gene, and the expression of the reporter gene is determined by the binding of the nuclease expressed in the cell to the target sequence and cleaving the reporter construct.

4. The method according to claim 1, wherein the reporter construct comprises a first reporter gene, a target sequence recognized by the nuclease, and a second reporter gene, wherein the expression of the second reporter gene is determined by the binding of the nuclease expressed in the cell to the target sequence and cleaving the reporter construct.

5. The method according to claim 4, wherein the second reporter gene is linked with the first reporter gene out of frame.

6. The method according to claim 4, wherein a stop codon is inserted upstream of the second reporter gene.

7. The method according to claim 4, wherein the third step of sorting the cells sorts the cells that express both of the first reporter gene and the second reporter gene.

8. The method according to claim 1, wherein the reporter construct comprises a first reporter gene, a target sequence recognized by the nuclease, a second reporter gene, and a third reporter gene, wherein the first reporter gene is expressed regardless of the binding of the nuclease to the target sequence and cleaving the reporter construct,

but the expression of the second reporter gene, the third reporter gene, or both is determined by the binding of the nuclease expressed in the cell to the target sequence and cleaving the reporter construct.

9. The method according to claim 8, wherein the second reporter gene and the third reporter gene is linked to the first reporter gene out of frame.

10. The method according to claim 8, wherein the second reporter gene and the third reporter gene are linked out of frame to each other.

11. The method according to claim 8, wherein the second reporter gene and the third reporter gene are linked in frame to each other.

12. The method according to claim 1, wherein the reporter gene encodes a protein that is selected from the group consisting of beta-galactosidase, β

-lactamase, TEV-protease, dihydrofolate reductase, luciferase, Renilla luciferase, Gaussia luciferase, selection marker, surface marker, fluorescent protein, and antibiotic resistance protein.

13. The method according to claim 1, wherein the nuclease expressed in the second step is expressed in the cell temporarily, or is expressed from the gene inserted in the genome of the cell.

14. The method according to claim 1, wherein the third step of sorting the cell is performed by fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS).

15. The method according to claim 1, wherein the second step and the third step are repeated twice or more.

16. The method according to claim 1, wherein the nuclease activity is examined through the third step of sorting the cells, which sorts the cells expressing the reporter gene and the cells not expressing the reporter gene.

17. The method according to of claim 1, wherein one or two or more reporter constructs are inserted in the second step.

18. A reporter construct for identifying, selecting, or enriching the cells, wherein a specific endogenous nucleotide sequence is cleaved by a specific nuclease or modified by such cleavage;

comprising a first reporter gene, a target sequence recognized by the nuclease, and a second reporter gene are comprised; and

wherein the expression of the second reporter gene is determined by the binding of the nuclease to the target sequence and cleaving the reporter construct.

25. The reporter construct according to claim 18, wherein a frame-shift mutation of reporter construct is generated when a target sequence is cleaved by a specific nuclease.

26. The reporter construct according to claim 18, wherein the reporter gene encodes a protein that is selected from the group consisting of beta-galactosidase, β

-lactamase, TEV-protease, dihydrofolate reductase, luciferase, Renilla luciferase, Gaussia luciferase, selection marker, surface marker, fluorescent protein, and antibiotic resistance protein.

27. A host cell, comprising one or more than two of the reporter construct of claim 18.

28. A system for monitoring nuclease activity, comprising one or two or more of the reporter construct of claim 18;

a host cell;

a nuclease-expressing construct, wherein the reporter construct, the nuclease-expressing construct, or both are already introduced in the host cell or prepared aside from the cells.

19. A reporter construct comprising a first reporter gene, a target sequence recognized by the nuclease, a second reporter gene, and a third reporter gene, wherein

the first reporter gene is expressed regardless of the binding of the nuclease to the target sequence and cleaving the reporter construct, however

the expression of the second reporter gene, third reporter gene, or both is determined by the binding of the nuclease expressed in the cell to the target sequence and cleaving the reporter construct.

20. The reporter construct according to claim 19, which is for identifying, selecting, or enriching the cells, wherein a specific endogenous nucleotide sequence is cleaved by a specific nuclease or modified by such cleavage.

21. The reporter construct according to claim 19, wherein the second reporter gene and the third reporter gene are linked to the first reporter gene out of frame.

22. The reporter construct according to claim 19, wherein the second reporter gene and the third reporter gene are linked out of frame to each other.

23. The reporter construct according to claim 19, wherein the second reporter gene and the third reporter gene are linked in frame to each other.

24. The reporter construct according to claim 23, wherein the second reporter gene and the third reporter gene are the different types of reporter genes.

29. The reporter construct according to claim 19, wherein a frame-shift mutation of reporter construct is generated when a target sequence is cleaved by a specific nuclease.

30. The reporter construct according to claim 19, wherein the reporter gene encodes a protein that is selected from the group consisting of beta-galactosidase, β

-lactamase, TEV-protease, dihydrofolate reductase, luciferase, Renilla luciferase, Gaussia luciferase, selection marker, surface marker, fluorescent protein, and antibiotic resistance protein.

31. A host cell, comprising one or more than two of the reporter construct of claim 19.

32. A system for monitoring nuclease activity, comprising one or two or more of the reporter construct of claim 19;

a host cell;

a nuclease-expressing construct, wherein the reporter construct, the nuclease-expressing construct, or both are already introduced in the host cell or prepared aside from the cells.