High throughput system for producing recombinant viruses using site-specific recombination

• US 20040185565A1
• Filed: 03/07/2003
• Published: 09/23/2004
• Est. Priority Date: 03/09/2002
• Status: Active Grant

• Current Assignee: Neurogenex Co., Ltd.
• Original Assignee: Neurogenex Co., Ltd.
• Inventors: Kim, Kyung-Jin, Seen, Dong-Seung, Choi, Eun-Wook, Kim, Do-Hui, Kim, Min-Sung, Son, Ho-Sun, Yi, Yong-Weon, Jung, Neon-C
• Granted Patent: US 7,319,001 B2
• Time in Patent Office: Days
• US Class Current: 435/456
• CPC Class Codes: C12N 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2710/14143 viral genome or elements thereof as genetic vector

Abstract

Disclosed are the methods for producing recombinant viruses using site-specific recombination in vitro. In the present invention, circular viral genomic DNAs are digested with restriction enzymes to generate a linear form viral genomic DNAs flanked by site-specific recombination sites, and then are subjected to site-specific recombination with the desired genomic materials flanked by site-specific recombination sites in vitro. According to the present invention, since the site-specific recombination mixture can be applied to host cells without further procedures of selecting the desired recombinant viral genomic DNAs, it is possible to obtain numerous recombinant viruses rapidly at the same time. Thus, the present invention can be used as a high throughput system for generating and screening hundreds or thousands of recombinant viruses.

Claims

1. A method for preparing expressive circular recombinant viral vectors, which comprises the steps of:

i) providing viral genomic DNA containing at least two site-specific recombination sites and at least one restriction enzyme recognition site;

ii) providing gene cassette containing desired genomic materials flanked with at least two site-specific sites; and

iii) reacting DNA fragment of i) and gene cassette of ii) in vitro.

2. The method for preparing expressive circular recombinant viral vectors as claimed in claim 1, wherein the viral genomic DNAs are digested with restriction enzymes at one or more sites to generate linear formed viral genomic DNAs.

3. The method for preparing expressive circular recombinant viral vectors as claimed in claim 2, wherein the restriction enzyme recognizes base sequences of CCTNAGG.

4. The method for preparing expressive circular recombinant viral vectors as claimed in claim 1, wherein the virus is baculovirus, poliomavirus, papilomavirus, or HBV.

5. A recombinant viral vector prepared according to any one of methods of claims 1 to 4.

6. A method of screening recombinant viruses containing desired genomic materials, which comprises the steps of:

i) providing viral genomic DNA fragment containing at least two site-specific recombination sites and at least one restriction enzyme recognition site;

ii) providing gene cassette containing desired genomic materials flanked with at least two site-specific sites;

iii) reacting DNA fragment of i) and gene cassette of ii) in vitro to produce reaction mixture including expressive circular recombinant viral vectors;

iv) transferring the reaction mixture of the iii) to host cells in multi-well plate; and

v) identifying the features expressed in host cells.

7. The method of screening recombinant viruses containing desired genomic materials, wherein the virus is baculovirus, poliomavirus, papilomavirus, or HBV.

8. A method of preparing recombinant viruses having circular genomic DNAs, which comprises the steps of:

i) providing viral genomic DNA fragment containing at least two site-specific recombination sites and at least one restriction enzyme recognition site;

ii) providing gene cassette containing desired genomic materials flanked with at least two site-specific sites;

iii) reacting DNA fragment of i) and gene cassette of ii) in vitro to produce reaction mixture including expressive circular recombinant viral vectors; and

iv) transferring the reaction mixture of the iii) to host cells in multi-well plate.

9. The method of preparing recombinant viruses having circular genomic DNAs as claimed in claim 8, wherein the viral genomic DNA fragment is non-infectious to animal cells or cannot replicate and cannot form viral particles, while includes bacterial replication origin and antibiotics resistance DNAs.

10. The method of preparing recombinant viruses having circular genomic DNAs as claimed in claim 8, wherein the transfer of the reaction mixture to host cells is carried out in multi-well plate.

11. The method of preparing recombinant viruses having circular genomic DNAs as claimed in claim 8, wherein viral genomic DNAs of i) are digested with restriction enzyme at one or more sites to generate linear formed viral genomic DNAs.

12. The method of preparing recombinant viruses having circular genomic DNAs as claimed in claim 11, wherein the restriction enzyme recognizes base sequences of CCTNAGG.

13. The method of preparing recombinant viruses having circular genomic DNAs as claimed in claim 8, wherein the virus is baculovirus, poliomavirus, papilomavirus or HBV.

14. A recombinant virus prepared according to any one of methods of claims 8-13.