Gene trap construct for identification and isolation of genes

• US 6,303,327 B1
• Filed: 09/08/1999
• Issued: 10/16/2001
• Est. Priority Date: 12/06/1996
• Status: Expired due to Fees

• Current Assignee: HOLZER, DIETER AND VON MELCHNER, HARALD
• Original Assignee: Dieter Holzer, Harald Von Melchner
• Inventors: Von Melchner, Harald, Hölzer, Dieter
• Primary Examiner(s): Achutamurthy, Ponnathapu
• Assistant Examiner(s): BRAGDON, KATHLEEN KERR
• Application Number:
  US09/319,495
• Time in Patent Office: 769 Days
• Field of Search: 536/23.1, 536/23.2, 435/320.1, 435/252.3, 435/254.11, 435/419, 435/325, 435/471, 435/18, 435/6
• US Class Current: 435/18
• CPC Class Codes: C12N 15/1051 Gene trapping, e.g. exon-, intron-, IRES-, signal sequence-trap cloning, trap vectors
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters

Abstract

The invention under consideration concerns a gene-trapping construct containing a first reporter gene which after activation can activate a second reporter gene, and the use of this gene-trapping construct for identification and isolation of genes, especially genes transiently expressed.

The invention under consideration furthermore concerns a cell, preferably a mammalian cell, containing the abovementioned gene-trapping construct. The invention under consideration in addition concerns the use of this mammalian cell for identification and/or isolation of genes, particularly transient genes. Furthermore the invention concerns a vector containing the abovementioned gene-trapping construct, as well as a kit for identification and/or isolation of genes, especially transient genes, that contains at least the abovementioned gene-trapping construct or the abovementioned vector.

In conclusion, the invention under consideration concerns a process for identification and/or isolation of genes, particularly transient genes.

Claims

1. A system for identifying a gene expressed in a cell, comprising:

a) a first nucleic acid sequence comprising a first reporter gene encoding a site-specific recombinase, wherein said first reporter gene is not under the control of a promoter; and

b) a second nucleic acid sequence comprising, in order from 5′

to 3′

,

i. a first promoter,

ii. a first target sequence for said recombinase,

iii. an intervening nucleotide sequence,

iv. a second target sequence for said recombinase, and

v. a second reporter gene,

wherein said first reporter gene, after being incorporated into an endogenous gene and being transcriptionally activated by an endogenous cellular promoter, is transcribed, and the encoded recombinase is produced, which encoded recombinase activates transcription of the second reporter gene, thereby allowing identification of the transcriptionally active gene under control of the endogenous cellular promoter.

2. The system of claim 1, characterized in that the recombinase is a Cre recombinase.

3. The system of claim 1, characterized in that the recombinase is a Flp recombinase.

4. The system of claim 1, characterized in that the second reporter gene encodes a protein.

5. The system of claim 4, characterized in that the protein is an IL-3.

6. The system of claim 4, characterized in that the protein is a β

-galactosidase.

7. The system of claim 1, characterized in that the first promoter is a phosphoglycerate kinase (pgk) promoter.

8. The system of claim 1, wherein the intervening nucleotide sequence comprises a selectable marker.

9. The system of claim 8, characterized in that the selectable marker encodes a protein that confers antibiotic resistance.

10. The system of claim 8, characterized in that the selectable marker encodes a thymidine kinase-neomycin phosphotransferase fusion protein.

11. The system of claim 8, characterized in that the selectable marker encodes a neomycin phosphotransferase.

12. The system of claim 1 wherein the first nucleic acid sequence and the second nucleic acid sequence are physically linked.

13. The system of claim 1, wherein the first nucleic acid sequence and the second nucleic acid sequence are not physically linked.

14. A vector comprising a system of claim 12.

15. A cell, comprising a system as claimed in any one of claims 1, 2, 3, 4, 5, or 6.

16. The cell of claim 15, characterized in that said cell is selected from the group consisting of a haemopoietic precursor cell and a totipotent embryonic stem cell.

17. A cell comprising a system as claimed in any one of claims 1, 2, 4, 5, or 6, wherein the cell is a mammalian cell, wherein the first nucleic acid sequence comprises a first reporter gene encoding a Cre recombinase, wherein the second nucleic acid sequence comprises, in order from 5′

to 3′

, a phosphoglycerate kinase (pgk) promoter, a first loxP target sequence, a nucleotide sequence encoding a thymidine kinase-neomycin transferase fusion protein (TKNeoPT), a second loxP target sequence, and a second reporter gene encoding an interleukin-3 (IL-3), wherein the Cre recombinase acts on the first and second loxp target sequences, resulting in deletion of the TKNeoPT-encoding nucleotide sequence such that the IL-3-encoding second reporter gene is brought under transcriptional control of the pgk promoter.

18. A method of identifying a transcriptionally active gene in a eukaryotic cell, comprising the steps of:

introducing a system as claimed in claim 1 into a eukaryotic cell in a population of cells;

selecting a cell from the population wherein the first reporter gene is incorporated into an endogenous gene under transcriptional control of an endogenous promoter; and

identifying the endogenous gene into which the first reporter gene is incorporated.

19. The method of claim 18, wherein the transcriptionally active gene is one that is transiently expressed in the cell.

20. The method of claim 19, wherein the transiently expressed gene is selected from the group consisting of a gene involved in programmed cell death, a cell cycle gene, a DNA repair gene, and a differentiation-specific gene.

21. The system of claim 1, wherein the first nucleic acid sequence comprises a first reporter gene encoding a Cre recombinase, wherein the second nucleic acid sequence comprises, in order from 5′

to 3′

, a phosphoglycerate kinase (pgk) promoter, a first loxP target sequence, a nucleotide sequence encoding a thymidine kinase-neomycin phosphotransferase fusion protein (TKNeoPT), a second loxP target sequence, and a second reporter gene encoding an interleukin-3 (IL-3), wherein the Cre recombinase acts on the first and second loxP target sequences, resulting in deletion of the TKNeoPT-encoding nucleotide sequence such that the IL-3-encoding second reporter gene is brought under transcriptional control of the pgk promoter.

22. A method of identifying a transcriptionally active gene in a cell, comprising:

a) introducing the system of claim 21 into an IL-3-dependent mammalian cell in a population of cells;

b) culturing the population of cells in a medium containing neomycin, thereby selecting a cell comprising the system;

c) culturing the cell selected in step (b) in IL-3-free medium, thereby selecting a cell in which the first reporter gene is integrated into a transcriptionally active endogenous gene; and

d) identifying the endogenous gene into which the first reporter gene is integrated.

23. The method of claim 22, wherein the IL-3-dependent mammalian cell is an FDCP-1 cell.

24. The method of claim 22, wherein the endogenous gene is one that encodes a gene product involved in programmed cell death.

25. The system of claim 1, wherein the first nucleic acid sequence comprises a first reporter gene encoding a Cre recombinase, wherein the second nucleic acid sequence comprises, in order from 5′

to 3′

, a phosphoglycerate kinase (pgk) promoter, a first recombinase target sequence, a nucleotide sequence encoding a thymidine kinase-neomycin phosphotransferase fusion protein (TKNeoPT), a second recombinase target sequence, and a second reporter gene encoding a β

-galactosidase, wherein the Cre recombinase acts on the first and second recombinase target sequences, resulting in deletion of the TKNeoPT-encoding nucleotide sequence such that the β

-galactosidase-encoding second reporter gene is brought under transcriptional control of the pgk promoter.

26. A method of identifying a gene transcriptionally activated during differentiation, comprising:

a) introducing the system of claim 25 into a totipotent embryonic stem cell in a population of totipotent embryonic stem cells;

b) culturing the population of cells in a medium containing neomycin, thereby selecting a cell that does not produce the Cre recombinase;

c) inducing the selected cell to differentiate, thereby activating a differentiation-specific gene;

d) selecting induced cells that produce β

-galactosidase, thereby selecting cells comprising the first reporter gene integrated into a cellular gene; and

e) identifying the cellular gene into which the first reporter gene is integrated.