Light-sensitive ion-passing molecules

Patent metadata

Field	Value
Publication number	US-9359449-B2
Application number	US-201213623612-A
Country	US
Kind code	B2
Filing date	Sep 20, 2012
Priority date	Mar 17, 2010
Publication date	Jun 7, 2016
Grant date	Jun 7, 2016

Abstract

The invention provides polynucleotides and methods for expressing light-activated proteins in animal cells and altering an action potential of the cells by optical stimulation. The invention also provides animal cells and non-human animals comprising cells expressing the light-activated proteins.

First claim

What is claimed is:

1. A method of identifying transsynaptic connection between neuronal cells in an animal or a tissue, comprising:
a) administering a first viral vector encoding a Cre recombinase fused to a transcellular tracer protein to neuronal cells in region A of the animal or tissue, wherein the transcellular tracer protein is wheat germ agglutinin (WGA) or tetanus toxin-fragment C (TTC), wherein the first viral vector is selected from the group consisting of an adenoassociated virus vector, a herpes simplex virus vector, and a lentiviral vector;
b) administering a second viral vector encoding a light-activated protein to neuronal cells in region B of the animal or tissue, wherein the expression of the light-activated protein depends on the presence of the Cre recombinase, wherein the light-activated protein comprises an amino acid sequence at least 95% identical to the sequence shown in SEQ ID NO:3, SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:4, wherein the second viral vector is selected from the group consisting of an adenoassociated virus vector, a herpes simplex virus vector, and a lentiviral vector; and
c) identifying neuronal cells expressing the light-activated protein in region B, wherein the expression of the light-activated protein in the neuronal cells indicates that these cells are in transsynaptic connection with the cells in region A.

2. The method of claim 1 , wherein the first viral vector and the second viral vector are lentiviral vectors.

3. The method of claim 1 , wherein the transcellular tracer protein is wheat germ agglutinin (WGA).

4. The method of claim 1 , wherein the light-activated protein comprises an amino acid sequence at least 95% identical to the sequence shown in SEQ ID NO:3.

5. A method of generating optical control of targeted neuronal cells in an animal or tissue, comprising:
a) administering a first viral vector expressing a Cre recombinase fused to a transcellular tracer protein to region A of the animal or tissue, wherein the transcellular tracer protein is wheat germ agglutinin (WGA) or tetanus toxin-fragment C (TTC), wherein the first viral vector is selected from the group consisting of an adenoassociated virus vector, a herpes simplex virus vector, and a lentiviral vector;
b) administering a second viral vector encoding a light-activated protein to region B of the animal or tissue, wherein the expression of the light-activated protein depends on the presence of the Cre recombinase, wherein the neuronal cells in region A and in region B are in transsynaptic connection, and wherein the light-activated protein comprises an amino acid sequence at least 95% identical to the sequence shown in SEQ ID NO:3, SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:4, wherein the second viral vector is selected from the group consisting of an adenoassociated virus vector, a herpes simplex virus vector, and a lentiviral vector; and
c) controlling action potential of a neuronal cell in region B with light that activates the light-activated protein.

6. The method of claim 5 , wherein the first and the second viral vectors are lentiviral vectors.

7. The method of claim 5 , wherein the transcellular tracer protein is wheat germ agglutinin (WGA).

8. The method of claim 5 , wherein the light-activated protein comprises an amino acid sequence at least 95% identical to the sequence shown in SEQ ID NO:3.

9. A method of controlling action potential of a neuron in an animal, comprising activating a light-activated protein in the neuron with light to generate action potential change, wherein expression of the light-activated protein in the neuron is generated by:
a) administering a first viral vector expressing a Cre recombinase fused to a transcellular tracer protein to region A of the animal, wherein the transcellular tracer protein is wheat germ agglutinin (WGA) or tetanus toxin-fragment C (TTC), wherein the first viral vector is selected from the group consisting of an adenoassociated virus vector, a herpes simplex virus vector, and a lentiviral vector; and
b) administering a second viral vector encoding a light-activated protein to region B of the animal which contains the neuron, wherein the expression of the light-activated protein depends on the presence of the Cre recombinase, wherein the neurons in region A and region B are in transsynaptic connection, wherein the light-activated protein comprises an amino acid sequence at least 95% identical to the sequence shown in SEQ ID NO:3, SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:4, wherein the second viral vector is selected from the group consisting of an adenoassociated virus vector, a herpes simplex virus vector, and a lentiviral vector.

10. The method of claim 9 , wherein the first viral vector and the second viral vector are lentiviral vectors.

11. The method of claim 9 , wherein the transcellular tracer protein is wheat germ agglutinin (WGA).

12. The method of claim 9 , wherein the light-activated protein comprises an amino acid sequence at least 95% identical to the sequence shown in SEQ ID NO:3.

13. The method of claim 1 , wherein the transcellular tracer protein is TTC.

14. The method of claim 1 , wherein the light-activated protein comprises:
a) a core amino acid sequence at least 95% identical to the sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; and
b) an endoplasmic reticulum (ER) export signal.

15. The method of claim 14 , wherein the ER export signal comprises the amino acid sequence FCEYENEV (SEQ ID NO:12).

16. The method of claim 14 , where the light-activated protein comprises a membrane trafficking signal.

17. The method of claim 16 , wherein the membrane trafficking signal comprises the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO:11).

18. The method of claim 5 , wherein the transcellular tracer protein is TTC.

19. The method of claim 5 , wherein the light-activated protein comprises:
a) a core amino acid sequence at least 95% identical to the sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; and
b) an endoplasmic reticulum (ER) export signal.

20. The method of claim 19 , wherein the ER export signal comprises the amino acid sequence FCEYENEV (SEQ ID NO:12).

21. The method of claim 19 , where the light-activated protein comprises a membrane trafficking signal.

22. The method of claim 16 , wherein the membrane trafficking signal comprises the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO:11).

23. The method of claim 9 , wherein the transcellular tracer protein is TTC.

24. The method of claim 9 , wherein the light-activated protein comprises:
a) a core amino acid sequence at least 95% identical to the sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; and
b) an endoplasmic reticulum (ER) export signal.

25. The method of claim 24 , wherein the ER export signal comprises the amino acid sequence FCEYENEV (SEQ ID NO:12).

26. The method of claim 24 , where the light-activated protein comprises a membrane trafficking signal.

27. The method of claim 26 , wherein the membrane trafficking signal comprises the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO:11).

Assignees

• Univ Leland Stanford Junior

Inventors

• Deisseroth Karl
• Zhang Feng
• Gradinaru Viviana

Classifications

• C12N2740/15043

  viral genome or elements thereof as genetic vector · CPC title

• A01K2267/0393

  Animal model comprising a reporter system for screening tests · CPC title

• C12N2799/025

  where the vector is derived from a parvovirus · CPC title

• C07K14/405 Primary

  from algae · CPC title

• C07K2319/02

  containing a signal sequence · CPC title