Process for site-directed integration of multiple copies of a gene in a mould
US6436643B1 · kind B1 · utility
Assignee
Inventors
Key dates
| Filing date | Aug 2, 2000 |
| Grant date | Aug 20, 2002 |
| Priority date | — |
| Expiry date | Aug 2, 2020 |
Classification
- Technology area (CPC C)Chemistry; Metallurgy
- CPC primaryC12N15/80
- WIPO fieldBiotechnology
- WIPO sectorChemistry
Abstract
A process for site-directed integration of multiple copies of a gene in a mould is provided, which comprises transforming a mould cell containing in its chromosomal DNA a restriction site for a rare-cutting endonuclease, e.g., I-Scel, preferably introduced at a desired locus, e.g., within a selectable marker gene or in the neighborhood thereof, with a piece of DNA comprising multiple copies of at least one expressible gene comprising at least one structural gene encoding a desired protein, surrounded by two DNA fragments homologous to part of the DNA upstream and downstream, and in the neighborhood, of said restriction site, while during the transformation of the mould the presence of the rare-cutting endonuclease is provided, followed by selecting or screening for a mould cell in which the multiple gene copies of said expressible gene are inserted into the chromosomal DNA of the mould. The piece of DNA can comprise a third DNA fragment completing any disrupted or partially deleted selectable marker gene in the chromosomal DNA. Preferably the mould belongs to the genus Aspergillus, especially to the species Aspergillus awamori. Also provided are a transformed mould obtainable by a …
Source: USPTO / EPO open patent data. Objective bibliographic and citation counts.