Optimization of cells for endogenous gene activation

• US 7,008,764 B1
• Filed: 12/01/1998
• Issued: 03/07/2006
• Est. Priority Date: 12/01/1997
• Status: Expired due to Term

• Current Assignee: Roche Diagnostics GmbH (Roche Holding AG)
• Original Assignee: Roche Diagnostics GmbH (Roche Holding AG)
• Inventors: Honold, Konrad, Stern, Anne, Holtschke, Thomas
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US09/203,500
• Time in Patent Office: 2,653 Days
• Field of Search: 435/6, 435/320.1, 435/455, 536/23.1, 536/24.1
• US Class Current: 435/6.13
• CPC Class Codes: C07K 14/505 Erythropoietin [EPO]
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/65 using markers enzymes used as markers C12N15/52
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: C12N 2830/002 inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters

Abstract

The invention concerns a process for optimizing the gene expression in cells. A first aspect concerns a process for changing the expression of a nucleic acid sequence which is present endogenously in a eukaryotic cell by introduction of a heterologous expression control sequence into the genome of the cell by means of homologous recombination as well as site-specific recombinase-mediated excision of inserted foreign DNA and its replacement by further heterologous expression control sequences or/and amplification genes. In addition the invention concerns the introduction of one or several nucleic acid sequences to which an activator protein or an activator protein complex binds e.g. a hypoxia-inducible factor (HIF), into the genome of a eukaryotic cell by homologous recombination in order to change the expression of a target gene. Furthermore the invention concerns a process for testing the influence of 5′ or 3′ non-coding nucleic acid fragments on the expression of a target gene by determining the expression of a reporter gene. In addition the invention concerns a process for providing a DHFR-negative eukaryotic cell containing a recombinase target sequence as well as the expression of a nucleic acid sequence inserted into the recombinase target sequence.

Claims

1. A process for changing the expression of a nucleic acid sequence which is present endogenously in a eukaryotic cell, the method comprising

(a) transfecting the cell with a vector comprising the following sequences

(i) at least one sequence, which upon expression is capable of changing the expression of the nucleic acid sequence which is present endogenously in the cell, and selected from the group consisting of a heterologous expression control sequence and an amplification gene,

(ii) a sequence encoding a positive selection marker,

(iii) at least two target sequences for a site-specific recombinase flanking the sequences of (i) and (ii), and

(iv) DNA sequences which flank the sequences of (i), (ii) and (iii) and are homologous to a nucleic acid section in the genome of the cell in order to allow a homologous recombination,

(b) culturing the transfected cell under conditions under which a homologous recombination of the vector takes place,

(c) isolating the cell obtained according to step (b), and

(d) expressing the at least one sequence of (i) to thereby change the expression of the nucleic acid sequence which is present endogenously in the cell.

2. The process as claimed in claim 1, wherein the site-specific recombinase target sequences are loxP sequences.

3. The process as claimed in claim 1, wherein the vector further comprises a negative selection marker gene which is located outside the homologous DNA sequences (iv).

4. The process as claimed in claim 1, further comprising, after step (d), cutting the sequences of (i) and (ii) flanked by the site-specific recombinase target sequences out of the genome of the cell by transient activation of a site-specific recombinase that recognizes the target sequences.

5. A vector suitable for homologous recombination, comprising the following sequences

(i) at least one sequence selected from the group consisting of an expression control sequence and an amplification gene each of which upon expression is capable of changing the expression of the nucleic acid sequence which is present endogenously in the cell,

(ii) a sequence encoding a positive selection marker,

(iii) at least two target sequences for a site-specific recombinase flanking the sequences of (i) and (ii), and

(iv) DNA sequences which flank the sequences of (i), (ii) and (iii) and are homologous to a nucleic acid section in the genome of a cell in order to allow a homologous recombination, and

(v) optionally a sequence encoding a negative selection marker.

6. A vector, comprising

(i) at least one sequence selected from the group consisting of a heterologous expression control sequence and an amplification gene each of which upon expression is capable of changing the expression of the nucleic acid sequence which is present endogenously in the cell,

(ii) a sequence encoding a positive selection marker,

(iii) at least two recombinase target sequences flanking the sequences of (i) and (ii), and

(iv) optionally a sequence encoding a negative selection marker.

7. A eukaryotic cell, comprising

(a) at least one chromosomally-located sequence selected from the group consisting of a heterologous expression control sequence and an exogenous amplification gene in operative linkage with a nucleic acid sequence which is present endogenously in the cell, and

(b) recombinase target sequences flanking the sequence (a).

8. The eukaryotic cell of claim 7, wherein the cell is a human cell.

9. A process for changing the expression of a nucleic acid sequence which is present endogenously in a eukaryotic cell, the method comprising

(a) transfecting the cell with a vector comprising

(i) at least one nucleic acid sequence which binds an activator protein,

(ii) a sequence encoding a positive selection marker, and

(iii) DNA sequences which flank the sequences of (i) and (ii) and are homologous to a nucleic acid section in the genome of the cell in order to allow a homologous recombination,

(b) culturing the transfected cell under conditions under which a homologous recombination of the vector takes place,

(c) isolating the cell obtained according to step (b), and

(d) expressing the sequence of (i) under conditions under which the activator protein is bound thereby changing the expression of the nucleic acid sequence which is present endogenously in the cell.

10. The process as claimed in claim 9, wherein the sequence (i) is a hypoxia-inducible factor-binding nucleic acid sequence.

11. A eukaryotic cell obtainable by the process as claimed in claim 9.

12. The eukaryotic cell of claim 11, wherein the cell is a human cell.

13. A vector suitable for homologous recombination, comprising

(i) at least one nucleic acid sequence which binds an activator protein,

(ii) a positive selection marker gene, and

(iii) DNA sequences which flank the sequences (i) and (ii) and are homologous to a nucleic acid section in the genome of a cell in order to allow a homologous recombination.

14. A eukaryotic cell, comprising at least one chromosomally-located exogenous nucleic acid sequence which binds an activator protein/activator protein complex which is operatively linked with a gene which is present endogenously in the cell.

15. The eukaryotic cell of claim 14, wherein the cell is a human cell.

16. A process for testing the influence of non-coding nucleic acid sequences from the region of a target gene present endogenously in a eukaryotic cell on its expression, the process comprising

(a) transfecting the cell with a vector comprising

(i) a heterologous expression control sequence which is active or can be activated in the cell and is operatively linked with a reporter gene, and

(ii) non-coding nucleic acid sequences on the 5′

-side and/or the 3′

-side from the region of the target gene,

(b) culturing the cell under conditions under which the expression control sequence is active, and

(c) measuring the expression of the reporter gene to determine the influence of the non-coding nucleic acid sequences on the expression of the target gene.

17. A process for obtaining a DHFR-negative eukaryotic cell, the process comprising

(a) transfecting a DHFR-positive cell with a first vector comprising

(i) at least one DHFR-negative target sequence for a site-specific recombinase;

(ii) DNA sequences which flank sequence (i) and are homologous to a DHFR nucleic acid sequence which is present endogenously in the cell in order to allow a homologous recombination,

(iii) optionally a sequence encoding a first positive selection marker, and

(iv) optionally a sequence encoding a negative selection marker,

(b) culturing the transfected cell under conditions under which a homologous recombination of the vector takes place thereby incorporating the DHFR-negative target sequence into the DHFR-positive cell to create a DHFR-negative cell, and

(c) isolating the cells obtained according to step (b) to obtain a DHFR-negative eukaryotic cell.

18. A process for obtaining a eukaryotic cell containing a nucleic acid sequence to be amplified and a heterologous DHFR gene, the process comprising

(a) obtaining a DHFR-negative eukaryotic cell by the process as claimed in claim 17, (b) transfecting the cell of step (a) with a second vector comprising

(i) a nucleic acid sequence coding for a DHFR,

(ii) a nucleic acid sequence to be amplified which codes for a protein in an expressible form,

(iii) optionally a sequence encoding a second positive selection marker, and

(iv) at least two recombinase target sequences flanking the sequences of (i), (ii) and (iii), if present,

(c) culturing the transfected cell under conditions under which the sequences of (i), (ii) and (iii), if present, are integrated into the recombinase target sequence that is already present in the genome of the cell, and

(d) isolating the cell obtained according to step (c) to obtain a eukaryotic cell containing a nucleic acid sequence to be amplified and a heterologous DHFR gene.

19. The process of claim 18, wherein the second positive selection marker gene differs from the first positive selection marker gene.

20. A vector, comprising

(i) a nucleic acid sequence coding for a DHFR,

(ii) a nucleic acid sequence to be amplified which codes for a protein in an expressible form,

(iii) optionally a sequence encoding a positive selection marker, and

(iv) at least two recombinase target sequences flanking the sequences of (i), (ii) and (iii), if present.

21. A vector suitable for homologous recombination, comprising

(i) optionally a sequence encoding a positive selection marker,

(ii) at least one recombinase target sequence which flanks the sequence of (i), if present,

(iii) DNA sequences which flank the sequences of (i), if present, and (ii) and which are homologous to a DHFR nucleic acid sequence which is present endogenously in a cell in order to allow a homologous recombination, and

(iv) optionally a sequence encoding a negative selection marker which is outside the homologous DNA sequences (iii).

22. A mammalian cell, comprising

(a) at least one inactivated endogenous nucleic acid sequence coding for a DHFR, and

(b) at least one recombinase target sequence which is integrated into the genome in the region of the sequence (a).

23. The mammalian cell of claim 22, wherein the cell is a human cell.

24. A mammalian cell, comprising a heterologous nucleic acid sequence in the region of an endogenous DHFR gene locus, the heterologous sequence comprising

(i) a nucleic acid sequence coding for a DHFR,

(ii) a nucleic acid sequence coding for a desired protein, and

(iii) at least one recombinase target sequence.