Mutated internal ribosomal entry site (IRES) for controlled gene expression
View Patent ↗The present invention relates to a nucleic acid molecule comprising one or multiple mutant IRES elements. Further, the present invention relates to methods of enhancing gene expression and to methods of differentially controlling expression of one or multiple gene(s) of interest. In addition, the present invention relates to a kit for studying interactions or any application requiring co-expression of multiple genes.
1. Nucleic acid molecule comprising a first internal ribosome entry site (IRES) sequence, wherein the first IRES sequence is a mutant IRES sequence and comprises a nucleotide sequence selected from the group consisting of any one of the nucleotides sequences set forth in SEQ ID Nos. 9-12, 19 and 21.
2. Nucleic acid molecule according to claim 1 , further comprising a nucleotide sequence encoding a gene of interest A, wherein the mutant IRES sequence and the nucleotide sequence encoding a gene of interest A are operably linked.
3. Nucleic acid molecule according to claim 2 , wherein the nucleic acid molecule further comprises a nucleotide sequence encoding a gene of interest B.
4. Nucleic acid molecule according to claim 3 , wherein the gene of interest B is operably linked to a second IRES sequence.
5. Nucleic acid molecule according to claim 3 , wherein the gene of interest B comprises a nucleic acid sequence encoding a light chain of an antibody.
6. Nucleic acid molecule according to claim 4 , wherein the nucleic acid molecule comprises a gene of interest C operably linked to a third IRES.
7. Nucleic acid molecule according to claim 6 , wherein the gene of interest C comprises a nucleic acid sequence encoding a heavy chain of an antibody.
8. Nucleic acid molecule according to claim 6 , wherein the nucleic acid molecule comprises one or more further genes of interest, each operably linked to an IRES sequence.
9. Nucleic acid molecule according to claim 8 , wherein the second and/or third and/or any further IRES sequence independently is a wild type IRES or a mutant IRES sequence that differs from a wild type IRES sequence at one or more ATG translation initiation site(s).
10. Nucleic acid molecule according to claim 9 , wherein the second and/or third and/or any further IRES sequence independently comprises of a nucleotide sequence selected from the group consisting of any one of the nucleotides sequences set forth in SEQ ID Nos. 9-12, 19 and 21.
11. Nucleic acid molecule according to claim 1 , wherein the mutant IRES sequence comprises a short DNA sequence which contains an additional out-of-frame ATG and has the capacity to form a hairpin structure.
12. Nucleic acid molecule according to claim 1 , wherein said nucleic acid molecule is a DNA molecule.
13. Nucleic acid molecule according to claim 12 , wherein said nucleic acid molecule comprises gene of interest A and the mutant IRES sequence and further comprises genes of interest B and C and second and third IRES sequences wherein the order of the elements on the coding strand is:
5′-second IRES-gene of interest B-mutant IRES-gene of interest A-third IRES-gene of interest C-3′.
14. Nucleic acid molecule according to claim 1 , wherein the gene of interest A encodes a selection marker.
15. Nucleic acid molecule according to claim 1 , wherein said nucleic acid molecule is comprised in a vector.
16. Nucleic acid molecule according to claim 2 , further comprising a promoter operably linked to the gene of interest A and the mutant IRES sequence.
17. A method of identifying cells that provide for enhanced gene expression comprising the steps of:
(i) constructing a cell-based expression system comprising a promoter, a gene encoding for a selection marker, one or more genes of interest different from the selection marker gene, and a mutant IRES sequence that comprises a nucleotide sequence selected from the group consisting of any one of the nucleotides sequences set forth in SEQ ID Nos. 9-12, 19 and 21, wherein the mutant IRES sequence is operably linked to the gene encoding for a selection marker but not the one or more genes of interest;
(ii) incubating the expression system under conditions that allow expression of the selection marker and the one or more genes of interest; and
(iii) selecting for the selection marker, thus identifying the cells that provide for enhanced gene expression.
18. The method according to claim 17 , wherein the order of the elements on the coding strand is
:5′-promoter-gene of interest-mutant IRES-selection marker-3′.
19. The method according to claim 17 , wherein the one or more genes of interest are operably linked to an IRES sequence, wherein said IRES sequence is a wildtype IRES sequence or a mutant IRES sequence that differs from a wild type IRES sequence at one or more ATG translation initiation site(s).