Expression elements
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) a methylation-free GC-rich element comprising at least 500 contiguous nucleotides from the promoter region of a ribosomal protein gene;
b) a heterologous promoter; and
c) a transcribable nucleic acid sequence adjacent said heterologous promoter wherein the transcribable nucleic acid sequence is transcribed from said heterologous promoter and the level of said transcription is enhanced by said element.
2 . The polynucleotide according to claim 1 , wherein said ribosomal protein gene is selected from the group consisting of RPSA, RPS2, RPS3, RPS3A, RPS4, RPS5, RPS6, RPS7, RPS8, RPS9, RPS10, RPS11, RPS12, RPS13, RPS14, RPS15, RPS15A, RPS16, RPS17, RPS18, RPS19, RPS20, RPS21, RPS23, RPS24, RPS25, RPS26, RPS27, RPS28, RPS29, RPS30, RPL3, RPL4, RPL5, RPL6, RPL7, RPL7A, RPL8, RPL9, RPL10, RPL10A, RPL11, RPL12, RPL13, RPL13A, RPL14, RPL15, RPL17, RPL18, RPL18A, RPL19, RPL21, RPL22, RPL23, RPL23A, RPL24, RPL26, RPL27, RPL27A, RPL28, RPL29, RPL30, RPL31, RPL32, RPL34, RPL35, RPL35A, RPL36, RPL36A, RPL37, RPL37ARPL38, RPL39, RPL41, RPLP0, RPLP1, RPLP2.
3 . The polynucleotide according to claim 1 , wherein said ribosomal protein gene is RPS3 or RPS11.
4 . The polynucleotide according to claims 1 , wherein said element comprises at least 1000 contiguous nucleotides from the promoter region of a ribosomal protein gene.
5 . The polynucleotide according to claim 1 , wherein said element comprises more than 3 kb 5′ untranslated sequence from said ribosomal protein gene.
6 . (canceled)
7 . The polynucleotide according to claim 1 , wherein said methylation-free GC-rich element is an extended methylation-free CpG island.
8 . (canceled)
9 . The polynucleotide according to claim 1 , wherein said ribosomal protein gene is a mammalian gene.
10 . The polynucleotide according to claim 9 , wherein said ribosomal protein gene is a murine gene.
11 . The polynucleotide according to claim 10 , wherein said methylation-free GC-rich element comprises the nucleotide sequence of SEQ ID NO:1.
12 . The polynucleotide according to claim 10 , wherein said methylation-free GC-rich element comprises the nucleotide sequence of SEQ ID NO:2.
13 . The polynucleotide according to claim 9 , wherein said ribosomal protein gene is a human gene.
14 . The polynucleotide according to claim 1 , wherein said heterologous promoter is a constitutive promoter.
15 . The polynucleotide according to claim 14 , wherein said constitutive promoter is selected from the group consisting of cytomegalovirus early/immediate promoter, SV40, EF-1α, Rous sarcoma virus (RSV) LTR and HIV2 LTR
16 . The polynucleotide according to claim 1 , wherein said heterologous promoter is a tissue-specific promoter.
17 . The polynucleotide according to claim 16 , wherein said heterologous promoter is a tumour-selective promoter.
18 . The polynucleotide according to claim 17 , wherein said 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.
19 . The polynucleotide according to claim 1 , wherein said 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, cytokine, a protein kinase, a soluble receptor, a membrane-bound receptor, and a blood clotting factor.
20 . A vector comprising the polynucleotide of claim 1 .
21 . The vector according to claim 20 , wherein the vector is a eukaryotic expression vector.
22 . An expression vector comprising:
a) a methylation-free GC-rich element comprising at least 500 contiguous nucleotides from the promoter region of a ribosomal protein gene;
b) a heterologous promoter; and
c) a multiple cloning site;
wherein a transcribable nucleic acid sequence inserted into said multiple cloning site and expression of said transcribable nucleic acid sequence from said heterologous promoter is enhanced by said element.
23 . A host cell comprising the polynucleotide according to claim 1 .
24 . The host cell according to claim 23 , wherein said cell is selected from the group consisting of CHO, NS0, BHK, HeLa, and HepG2.
25 . A method of increasing expression of a polypeptide encoded by a transcribable nucleic acid comprising inserting the expression vector according to claim 22 into an appropriate host cell and culturing said host cell under suitable conditions, thereby to increase expression of said polypeptide.
26 . The method according to claim 25 , wherein said polypeptide is a therapeutically useful polypeptide.
27 . (canceled)
28 . The polynucleotide according to claim 1 , wherein the heterologous promoter is a guinea pig CMV immediate/early promoter.
29 . The polypeptide of claim 25 , wherein the polypeptide is an antibody or a functional epitope-binding fragment thereof.
30 . A method of increasing the expression of a transcribable nucleic acid in a host cell, the method comprising the steps of:
a) transfecting the host cell with a vector comprising the transcribable nucleic acid operably linked to a heterologous promoter which is operably linked to a methylation-free GC-rich element derived from an rps3 gene;
b) culturing the host cell under suitable conditions, thereby to allow for the expression of the transcribable nucleic acid,
wherein the expression of the transcribable nucleic acid is increased in the presence of the methylation-free GC-rich element relative to expression obtained in the presence of the heterologous promoter alone.
31 . The method of claim 30 , wherein the transcribable nucleic acid encodes an antibody.
32 . The method of claim 30 , wherein the rps3 gene is a murine gene.
33 . The method of claim 32 , wherein the murine gene is a mouse gene.
34 . The method of claim 32 , wherein the host cell is a CHO cell.
35 . The method of claim 32 , wherein the heterologous promoter is a guinea pig CMV promoter.