Processing

Please wait...

Settings

Settings

Goto Application

1. WO2006055836 - IN VIVO ALTERATION OF CELLULAR DNA

Note: Text based on automatic Optical Character Recognition processes. Please use the PDF version for legal matters

[ EN ]
What is claimed is:

1. A method of in vivo serial alteration of a nucleic acid sequence of interest in a target cell comprising the steps of:
a. contacting a sample of one or more target cells with a first exogenous nucleic acid sequence having a selection marker, and allowing the first exogenous nucleic acid sequence to exchange with a nucleic acid sequence in target cells in the sample to produce first recombinant target cells;
b. contacting the sample of one or more target cells resulting from step (a) with a first selection substrate, wherein the first recombinant target cells are selected if they express the selection marker to produce a sample of first selected recombinant target cells;
c. contacting the sample of one or more target cells resulting from step (b) with a second exogenous nucleic acid sequence, and allowing the second exogenous nucleic acid sequence to exchange with a nucleic acid sequence in the one or more first selected recombinant target cells to produce a sample of one or more further recombinant target cells; and
d. contacting the sample of one or more target cells resulting from step (c) with a second selection substrate, wherein the one or more further recombinant target cells are selected if they lack expression of the selection marker, thereby providing a sample of further recombinant target cells.

2. The method of claim 1, wherein the selection marker comprises a single selection component.

3. The method of claim 2, wherein the selection component is thy A.

4. The method of claim 1, wherein the selection marker comprises a positive selection component and a negative selection component.

5. The method of claim 4, wherein the positive selection component is a member selected from the group consisting of a visually detectable marker, a marker that allows growth in media lacking a nutrient, and a marker that confers resistance to a compound.

6. The method of claim 4, wherein the negative selection component is a visually detectable marker or a marker that is lethal to the cell in the presence of substrate.

7. The method of claim 1, wherein the first selection substrate comprises a minimal medium, a medium that lacks one or more nutrients essential to the one or more target cells or an antibiotic.

8. The method of claim 1, wherein the one or more target cells are altered by addition to their genome of one or more nucleotides.

9. The method of claim 1, wherein the one or more target cells are altered by removal of one or more nucleotides from their genome.

10. The method of claim 1, wherein the one or more target cells are altered by exchange of one or more nucleotides in their genome.

11. The method of claim 1, wherein the one or more target cells are altered by one or more of addition to their genome of one or more nucleotides, removal of one or more nucleotides from their genome and exchange of one or more nucleotides in their genome.

12. The method of claim 1, wherein alteration of the genome of the one or more target cells is made to one or more of an endogenous gene, an endogenous intergenic nucleic acid sequence, and a non-geneic nucleic acid sequence.

13. The method of claim 1, wherein an amino acid specificity of a tRNA synthetase is altered.

14. The method of claim 11, wherein the amino acid specificity of a tRNA synthetase is altered to utilize a different amino acid.

15. The method of claim 11, wherein a novel tRNA synthetase is expressed in the cell.

16. The method of claim 11, wherein an exogenous tRNA is expressed in the cell.

17. The method of claim 1, wherein a restriction endonuclease cleavage site is altered.

18. The method of claim 1, wherein the target cell is selected from the group consisting of a bacterial cell, a yeast cell, a plant cell, an insect cell, an amphibian cell, a nematode cell and a mammalian cell.

19. A method of in vivo serial alteration of a nucleic acid sequence of interest in a target cell comprising the steps of:
a. contacting a sample of one or more target cells with a first exogenous nucleic acid sequence having a first selection marker and a second selection marker, and allowing the first exogenous nucleic acid sequence to exchange with a nucleic acid sequence in target cells in the sample to produce first recombinant target cells;
b. contacting the sample of one or more target cells resulting from step (a) with a first selection substrate, wherein the first recombinant target cells are selected if they express the first selection marker to produce a sample of first selected recombinant target cells;
c. contacting the sample of one or more target cells resulting from step (b) with a second exogenous nucleic acid sequence, and allowing the second nucleic acid sequence to exchange with a nucleic acid sequence in the one or more first selected recombinant target cells to produce one or more further recombinant target cells; and
d. contacting the sample of one or more target cells resulting from step (c) with a second selection substrate, wherein the one or more further recombinant target cells are selected if they lack expression of the second selection marker, thereby providing a sample of selected further recombinant target cells.

20. A method of in vivo serial alteration of two or more nucleic acid sequences of interest in a target cell comprising the steps of:
a. contacting a sample of one or more target cells with a first exogenous nucleic acid sequence having a first selection marker and a second exogenous nucleic acid sequence having a second selection marker, and allowing the first and second exogenous nucleic acid sequences to exchange with corresponding nucleic acid sequences in target cells in the sample to produce first recombinant target cells; and
b. contacting the sample of one or more target cells resulting from step (a) with a first selection substrate and a second selection substrate, wherein the first recombinant target cells are selected if they express the first selection marker and the second selection marker to produce a sample of first selected recombinant target cells.

21. A method of in vivo serial alteration of a nucleic acid sequence of interest in a target cell comprising the steps of:
a. contacting a sample of one or more target cells simultaneously or in series with one or more exogenous nucleic acid sequences having one or more selection markers, and allowing each exogenous nucleic acid sequence to exchange with a nucleic acid sequence in target cells in the sample to produce recombinant target cells;
b. contacting the sample of one or more target cells resulting from step (a) with one or more selection substrates, wherein the recombinant target cells are selected on the basis of positive or negative selection.

22. A method of in vivo serial alteration of a nucleic acid sequence of interest in a target cell comprising the steps of:
a. contacting a sample of one or more target cells with a first exogenous nucleic acid sequence having a selection marker, and allowing the first exogenous nucleic acid sequence to exchange with a nucleic acid sequence in target cells in the sample to produce first recombinant target cells and non-altered target cells;
b. contacting the sample of one or more target cells resulting from step (a) with a first selection substrate, wherein the recombinant target cells survive if they express the selection marker to produce a sample including surviving recombinant target cells and non-altered target cells die if they lack expression of the selection marker;
c. contacting the sample of one or more target cells resulting from step (b) with a second exogenous nucleic acid sequence, and allowing the second exogenous nucleic acid sequence to exchange with a nucleic acid sequence in the surviving recombinant target cells to produce a sample including further recombinant target cells and non-altered target cells; and d. contacting the sample of one or more target cells resulting from step (c) with a second selection substrate, wherein the further recombinant target cells survive if they lack expression of the selection marker, and non-altered target cells die of they express the selection marker thereby providing a sample of living further recombinant target cells.