Methods for Live Imaging of Cells

• US 20140364333A1
• Filed: 06/27/2014
• Published: 12/11/2014
• Est. Priority Date: 03/15/2013
• Status: Abandoned Application

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: WU, Chao-ting, KIM, Caroline
• Application Number:
  US14/317,169
• Publication Number:
  US 20140364333A1
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 2565/619 being a video camera

Abstract

The present invention relates to methods of hybridizing nucleic acid probes to genomic DNA.

Claims

1. A method of imaging a live cell by fluorescence in situ hybridization comprising

combining the live cell under growth conditions with a labeled probe having a sequence complementary to a genomic nucleic acid sequence, and

imaging the labeled probe within the live cell bound to genomic DNA.

2. The method of claim 1 wherein the probe is a labeled locked nucleic acid probe, a labeled oligonucleotide, an ECHO probe, a molecular beacon, a labeled toe-hold probe, a labeled TALE or a labeled Cas9/RNA complex.

3. The method of claim 1 further including the step of removing unhybridized probe from within the live cell.

4. The method of claim 1 further including the step of removing unhybridized probe from within the live cell by placing the cell in probe-free growth media and allowing the cell to double once or twice.

5. The method of claim 1 further including the step of removing unhybridized probe from within the live cell by centrifugation of the cell and resuspension in probe-free growth media.

6. The method of claim 1 wherein the genomic nucleic acid sequence is undergoing replication.

7. The method of claim 1 wherein the genomic nucleic acid sequence is undergoing naturally occurring replication.

8. The method of claim 1 wherein replication of the genomic nucleic acid sequence is induced before being contacted by the labeled probe.

10. A method of imaging a live cell by fluorescence in situ hybridization comprising

placing the live cell under growth conditions;

synthesizing a Cas9 within the cell,

synthesizing RNA within the cell to bind genomic DNA and to complex with the Cas9 forming a Cas9/RNA complex,

labeling the Cas 9/RNA complex, and

imaging the labeled Cas 9/RNA complex within the live cell bound to genomic DNA.

11. The method of claim 10 wherein the Cas9 is synthesized in vivo by using an integrated construct, a transiently transfected construct, by injection into the cell of a syncitia of nuclei or via electroporation into cells and/or nuclei.

12. The method of claim 10 wherein the RNA is synthesized in vivo by using an integrated construct, a transiently transfected construct, by injection into the cell of a syncitia of nuclei or via electroporation into cells and/or nuclei.

13. The method of claim 10 wherein the Cas9/RNA complex is labeled by making a fusion protein that includes Cas9 and a reporter, by injection of RNA that has been attached to a reporter into the cell or by a syncitia of nuclei including RNA that has been attached to a reporter, by electroporation into cells or nuclei or by indirect labeling of the RNA by hybridization with a labeled secondary oligonucleotide.

14. The method of claim 10 wherein the label is a conditional reporter.

15. The method of claim 10 wherein the label is a conditional reporter based on the binding of Cas9/RNA to the target nucleic acid.

16. The method of claim 10 wherein the label is quenched and is then activated upon the Cas9/RNA complex binding to the target nucleic acid.

17. A method of imaging a live cell by fluorescence in situ hybridization comprising

genetically altering the live cell to include one or more nucleic acid sequences complementary to a probe, combining the live cell under growth conditions with the probe having a sequence complementary to the one or more sequences added, and

imaging the probe within the live cell bound to one or more sequences added.

18. A method of making a live cell for in situ hybridization comprising

genetically altering the live cell to include one or more nucleic acid sequences complementary to a probe.