Methods for gene disruption and uses thereof

• US 20030082591A1
• Filed: 07/24/2002
• Published: 05/01/2003
• Est. Priority Date: 07/24/2001
• Status: Abandoned Application

• Current Assignee: Affinium Pharmaceuticals, Inc.
• Original Assignee: Affinium Pharmaceuticals, Inc.
• Inventors: Awrey, Donald, Greenblatt, Jack
• Application Number:
  US10/202,442
• Publication Number:
  US 20030082591A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome

Abstract

The present invention relates to compositions and methods for in-frame disruption of a gene sequence by homologous recombination. The present invention may be used in certain embodiments to disrupt a gene without causing any downstream effects on non-target sequences. In certain embodiments, the inventive methods may be used to identify and/or characterize products encoded by essential genes, conditionally essential genes, and non-essential genes.

Claims

1. A method for disrupting a gene of an organism without introducing downstream effects, comprising:

(a) providing a host cell of an organism, wherein the host cell is capable of homologous recombination;

(b) identifying an open reading frame for a gene of the organism;

(c) introducing into the host cell a targeting polynucleotide comprising (i) a molecular tag and (ii) flanking homology clamps for aligning the targeting polynucleotide in-frame with the open reading frame of the gene; and

(d) selecting host cells having an in-frame disruption of the gene.

2. The method of claim 1, wherein the host cell comprises an endogenous recombination system.

3. The method of claim 1, wherein the host cell is expressing the exo, bet and gam genes from phage λ

.

4. The method of claim 1, which further comprises passage of the cells.

5. The method of claim 1, further comprising characterizing the essentiality of the gene by determining whether the in-frame insertion of the targeting polynucleotide into the genome of the host cell produced a change in the phenotype of the host cell attributable to disruption of the gene in the absence of any downstream effects.

6. The method of claim 5, wherein the change in the phenotype of the host cell is a change in the growth rate or viability of the host cell.

7. The method of claim 6, wherein a change in the growth rate or viability of the host cell is determined using PCR or DNA microarray analysis.

8. The method of claim 5, wherein the change in the phenotype of the host cell occurs under an environmental condition selected from the group consisting of oxygen tension, osmolarity, pH, temperature, nutrient availability, prolonged stationary phase, presence of a test compound, conditions encountered in a disease state, and combinations thereof.

9. The method of claim 1, wherein the molecular tag is inserted into the genome of the host cell under the control of the transcriptional, translational, or transcriptional and translational regulatory sequences of the disrupted gene.

10. The method of claim 1, wherein host cells having in-frame disruptions are selected by PCR with one primer that hybridizes to a region within the targeting polynucleotide insertion and one primer that hybridizes to a region of the genome of the host cell outside of the disrupted gene sequence.

11. The method of claim 1, wherein the molecular tag is a reporter gene encoding for a selectable or detectable marker.

12. The method of claim 11, wherein host cells having in-frame disruptions are selected based on expression of the reporter gene.

13. The method of claim 1, wherein the host cell is haploid.

14. The method of claim 1, wherein the host cell is diploid.

15. The method of claim 14, wherein disruption of one copy of a gene produces a change in the phenotype of the host cell.

16. The method of claim 1, wherein the host cell is prokaryotic.

17. The method of claim 16, wherein the host cell is selected from the group consisting of bacteria and archaebacteria.

18. The method of claim 1, wherein the host cell is eukaryotic.

19. The method of claim 18, wherein the eukaryotic cell is selected from the group consisting of algae, yeast, fungus, plant, insect, reptile, amphibian, fish, bird, rodent, mammal, monkey and human.

30. A database comprising the identity of essential genes for an organism wherein substantially all of the genes have been disrupted in-frame using the method of claim 1.

20. A method for characterizing the essentiality of a plurality of genes from an organism by in-frame gene disruption, comprising:

(a) providing a host cell of an organism, wherein the host cell is capable of homologous recombination;

(b) identifying at least five open reading frames for at least five genes of the organism;

(c) introducing into the host cell a targeting polynucleotide for each of the genes comprising (i) a molecular tag and (ii) flanking homology clamps for aligning the targeting polynucleotides in-frame with the open reading frames of the genes;

(d) selecting host cells having in-frame disruptions of the genes; and

(e) determining whether the in-frame insertions of the targeting polynucleotides into the genome of the host cell produced a change in the phenotype of the host cell attributable to disruption of the genes in the absence of any downstream effects.

21. The method of claim 20, wherein at least 25 genes are targeted for disruption by at least 25 targeting polynucleotides.

22. The method of claim 21, wherein at least 50 genes are targeted for disruption by at least 50 targeting polynucleotides.

23. The method of claim 22, wherein at least 100 genes are targeted for disruption by at least 100 targeting polynucleotides.

24. The method of claim 23, wherein at least 500 genes are targeted for disruption by at least 500 targeting polynucleotides.

25. The method of claim 20, wherein the at least 5 targeting polynucleotides are introduced into a single host cell culture.

26. The method of claim 20, wherein the at least 5 targeting polynucleotides are introduced into separate cultures of host cells.

27. The method of claim 20, wherein the sequence for each of the at least 5 molecular tags is the same.

28. The method of claim 20, wherein the sequence for each of the at least 5 molecular tags is different.

29. The method of claim 20, wherein all of the at least 5 targeting polynucleotides are introduced into the genome in-frame.