Expression vector containing a drug-resistance gene having a destabilizing sequence as selection marker
The present invention provides a process for preparing a retrovirus to be expressed at a high titer by specifically transferring a desired foreign gene into target cells. A pseudotyped retrovirus vector having a high titer can be prepared by transferring a DNA construction wherein a promoter, a loxP sequence, a VSV-G gene and a polyA addition signal are arranged in this order is transferred into cells carrying the retrovirus gag and pol gene expression systems, and then transferring a retrovirus vector containing the desired foreign gene thereinto, followed by the treatment with a recombinase.
1. An expression vector comprising a promoter, a recombinase recognition sequence, a selectable drug-resistance gene having an mRNA-destabilizing sequence, a polyA addition signal, a recombinase recognition sequence, a gene encoding a viral structural protein, and a polyA addition signal, arranged in this order, which produces a short-lived transcript of the drug-resistance gene and wherein said promoter transcribes the gene encoding a viral structural protein in a prepackaging cell.
2. The expression vector as set forth in claim 1 , in which the mRNA-destabilizing sequence is an mRNA-destabilizing sequence of a c-fos gene.
3. The expression vector as set forth in claim 1 , in which the drug-resistance gene is selected from the group consisting of a neomycin resistance gene, a puromycin resistance gene and a hygromycin resistance gene.
4. Cells into which the expression vector as set forth in claim 1 has been transferred and selected with the drug.
5. A process for producing cells expressing a gene encoding a viral structural protein in the expression vector as set forth in claim 1 , comprising:
(a) transferring the expression vector into cells,
(b) selecting cells which express the drug-resistance gene from the transferred expression vector, and
(c) expressing the gene encoding a viral structural protein in the expression vector in the selected cells.
6. A process for expressing a gene encoding a viral structural protein in the expression vector as set forth in claim 1 , comprising:
(a) transferring the expression vector into cells having gag and pol genes of a retrovirus,
(b) selecting prepackaging cells which express the drug-resistance gene from the transferred expression vector, and
(c) expressing the gene encoding a viral structural protein in the expression vector in the selected prepackaging cells.
7. An expression vector comprising a promoter, a recombinase recognition sequence, a selectable drug-resistance gene having an mRNA-destabilizing sequence, a polyA addition signal, a recombinase recognition sequence, a foreign gene, and a polyA addition signal, arranged in this order, which produces a short-lived transcript of the drug-resistance gene and wherein said promoter transcribes the foreign gene in a prepackaging cell.
8. The expression vector as set forth in claim 7 , in which the mRNA-destabilizing sequence is an mRNA-destabilizing sequence of a c-fos gene.
9. The expression vector as set forth in claim 7 , in which the drug-resistance gene is selected from the group consisting of a neomycin resistance gene, a puromycin resistance gene and a hygromycin resistance gene.
10. Cells into which the expression vector as set forth in claim 7 has been transferred and selected with the drug.
11. A process for producing cells expressing a foreign gene in the expression vector as set forth in claim 7 , comprising:
(a) transferring the expression vector into cells,
(b) selecting cells which express the drug-resistance gene from the transferred expression vector, and
(c) expressing the foreign gene in the expression vector in the selected cells.
12. A process for expressing a foreign gene in the expression vector as set forth in claim 7 , comprising:
(a) transferring the expression vector into cells having gag and pol genes of a retrovirus,
(b) selecting prepackaging cells which express the drug-resistance gene from the transferred expression vector, and
(c) expressing the foreign gene in the expression vector in the selected prepackaging cells.
13. An expression vector to be expressed in a prepackaging cell comprising a first LTR of a retrovirus genome and a packaging signal, a recombinase recognition sequence, a selectable drug-resistance gene having an mRNA-destabilizing sequence, a polyA addition signal, a recombinase recognition sequence, a foreign gene, and a second LTR of a retrovirus genome, arranged in this order, which produces a short-lived transcript of the drug-resistance gene.
14. The expression vector as set forth in claim 13 , in which the mRNA-destabilizing sequence is an mRNA-destabilizing sequence of a c-fos gene.
15. The expression vector as set forth in claim 13 , in which the drug-resistance gene is selected from the group consisting of a neomycin resistance gene, a puromycin resistance gene and a hygromycin resistance gene.
16. Cells into which the expression vector as set forth in claim 13 has been transferred and selected with the drug.
17. A process for producing cells expressing a foreign gene in the expression vector as set forth in claim 13 , comprising:
(a) transferring the expression vector into cells,
(b) selecting cells which express the drug-resistance gene from the transferred expression vector, and
(c) expressing the foreign gene in the expression vector in the selected cells.
18. A process for expressing a foreign gene in the expression vector as set forth in claim 13 , comprising:
(a) transferring the expression vector into cells having gag and pol genes of a retrovirus,
(b) selecting prepackaging cells which express the drug-resistance gene from the transferred expression vector, and
(c) expressing the foreign gene in the expression vector in the selected prepackaging cells.