Expression elements
View Patent ↗The invention relates to genetic elements capable of improving the levels of expression of operably-linked transcription units. In particular, said genetic elements are derived from the 5′ untranslated regions of ribosomal protein genes and may comprise a CpG island. Also provided are vectors and host cells comprising said genetic elements and methods of use to obtain high levels of recombinant gene expression.
1. An isolated polynucleotide comprising:
a. an extended methylation-free CpG island comprising at least 300 contiguous nucleotides from the promoter region of a mammalian rps3 gene;
b. a heterologous promoter; and
c. a transcribable nucleic acid sequence adjacent the heterologous promoter,
wherein the extended methylation-free CpG island is capable of increasing transcription of the transcribable nucleic acid sequence from the heterologous promoter relative to transcription in the absence of the extended methylation-free CpG island.
2. A eukaryotic expression vector comprising:
a. an extended methylation-free CpG island from the promoter region of a mammalian rps3 gene;
b. a heterologous promoter; and
c. a multiple cloning site,
wherein a transcribable nucleic acid sequence inserted into the multiple cloning site is capable of being transcribed from the heterologous promoter and the level of transcription is enhanced by the extended methylation-free CpG island relative to transcription in the absence of the extended methylation-free CpG island.
3. The polynucleotide according to claim 1 , wherein the extended methylation-free CpG island comprises at least 500 contiguous nucleotides from the promoter region of the mammalian rps3 gene.
4. The polynucleotide according to claim 1 , wherein the extended methylation-free CpG island comprises at least 1000 contiguous nucleotides from the promoter region of the mammalian rps3 gene.
5. The polynucleotide according to claim 1 , wherein the extended methylation-free CpG island further comprises one or more exons of the mammalian rps3 gene.
6. The polynucleotide according to claim 1 , wherein the mammalian rps3 gene is a human rps3 gene.
7. The polynucleotide according to claim 1 , wherein the mammalian rps3 gene is a rodent rps3 gene.
8. The polynucleotide according to claim 7 , wherein the rodent rps3 gene is a mouse rps3 gene.
9. The polynucleotide according to claim 8 , comprising the nucleotide sequence of SEQ ID NO:1.
10. The polynucleotide according to claim 1 , wherein the heterologous promoter is a constitutive promoter.
11. The polynucleotide according to claim 10 , wherein the constitutive promoter is selected from the group consisting of cytomegalovirus early/immediate promoter, SV40, EF-1α, Rous sarcoma virus (RSV) LTR and HIV2 LTR.
12. The polynucleotide according to claim 11 , wherein the constitutive promoter is a cytomegalovirus early/immediate promoter.
13. The polynucleotide according to claim 12 , wherein the constitutive promoter is a guinea pig cytomegalovirus early/immediate promoter.
14. The polynucleotide according to claim 12 , wherein the constitutive promoter is a mouse cytomegalovirus early/immediate promoter.
15. The polynucleotide according to claim 1 , wherein the heterologous promoter is a tissue-selective promoter.
16. The polynucleotide according to claim 15 , wherein the heterologous promoter is a tumour-selective promoter.
17. The polynucleotide according to claim 16 , wherein the promoter is selected from the group consisting of carcino-embryonic antigen (CEA), prostate-specific antigen (PSA), cyclooxygenase-2 (COX-2), alpha-fetoprotein (AFP), tyrosinase, and T-cell Factors 1-4 (TCF) based promoters.
18. The polynucleotide according to claim 1 , wherein the transcribable nucleic acid encodes a polypeptide selected from the group consisting of an antibody, a functional epitope-binding fragment of an antibody, a growth factor, a cytokine, a protein kinase, a soluble receptor, a membrane-bound receptor and a blood clotting factor.
19. A vector comprising of the polynucleotide of claim 18 .
20. The vector according to claim 19 , wherein the vector is eukaryotic expression vector.
21. A host cell comprising the polynucleotide of claim 1 or the vector of claim 2 .
22. The host cell according to claim 21 , wherein the cell is selected from the group consisting of CHO, NS0, BHK, HeLa and HepG2.
23. A method of increasing the percentage of host cells expressing a polypeptide encoded by a polynucleotide with which said cells are transfected comprising:
a. transfecting a population of cells with an isolated polynucleotide according to claim 1 , wherein the transcribable polynucleotide encodes a polypeptide; and
b. measuring the number of host cells which express the polypeptide, wherein an increase in the number of cells expressing the polypeptide relative to the cells expressing the polypeptide by a polynucleotide lacking an extended methylation-free CpG island is an indication that the percentage of host cells expressing the polypeptide is increased.
24. The method according to claim 23 , wherein the polypeptide is an antibody or a functional epitope-binding fragment thereof.
25. The eukaryotic expression vector according to claim 2 , wherein the transcribable nucleic acid encodes an antibody or a functional epitope-binding fragment of an antibody.
26. A method of expressing a polypeptide encoded by a transcribable nucleic acid comprising inserting an expression vector according to claim 2 or claim 25 into an appropriate host cell and culturing the host cell under conditions to allow expression of the polypeptide.
27. An isolated polynucleotide comprising:
a. an extended methylation-free CpG island comprising the nucleotide sequence set forth in SEQ ID NO:1;
b. a heterologous promoter; and
c. a transcribable nucleic acid sequence adjacent the heterologous promoter,
wherein the transcribable nucleic acid sequence is transcribed from the heterologous promoter and transcription is enhanced by the extended methylation-free CpG island relative to transcription in the absence of the extended methylation-free GpG island.