Compositions and methods for generating expression vectors through site-specific recombination

• US 6,551,828 B1
• Filed: 06/28/2000
• Issued: 04/22/2003
• Est. Priority Date: 06/28/2000
• Status: Expired due to Fees

• Current Assignee: Protemation, Inc.
• Original Assignee: Protemation, Inc.
• Inventors: Clark, Robin
• Primary Examiner(s): Wang, Andrew
• Assistant Examiner(s): Friend, Tomas
• Application Number:
  US09/606,323
• Time in Patent Office: 1,028 Days
• Field of Search: 435/5, 435/7.31, 435/7.21, 435/7.32, 435/471, 435/455, 435/462, 935/28
• US Class Current: 435/462
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease

Abstract

Compositions, kits, and methods are provided for use in a recombinational cloning or subcloning methods for constructing expression vectors which comprise: ligating a library of double-stranded linear donor DNAs, where each member of the library includes a donor DNA sequence, with a double-stranded linear driver DNA which includes a promoter sequence and a donor recombination site to form a single circular donor DNA, the single circular donor DNA not including an origin of replication, where the donor DNA sequence is under the transcriptional control of the promoter; and contacting the circular donor DNA and a circular acceptor acceptor vector in the presence of a recombinase to form a single fused circular vector, the circular acceptor vector comprising an origin of replication and an acceptor recombination site capable of recombining with the circular donor DNA.

Claims

1. A method for generating a recombinant expression vector for expressing a donor DNA sequence, comprising:

contacting a circular double-stranded donor DNA and a circular acceptor vector in the presence of a recombinase under conditions suitable for the circular double-stranded donor DNA and the circular acceptor vector to recombine to form a single fused circular vector that combines sequences of both the circular double-stranded donor DNA and the circular acceptor vector, thereby forming the recombinant expression vector for expressing the donor DNA sequence, wherein

the circular double-stranded donor DNA comprises a donor DNA sequence, a single donor recombination site, and at least one selectable marker, but not including an origin of replication;

the circular acceptor vector comprises an origin of replication and a single acceptor recombination site capable of recombining with the circular donor DNA; and

the recombinase recognizes the donor and acceptor recombination sites and catalyzes fusion of the whole sequence of the circular double-stranded donor DNA into the circular acceptor vector.

2. The method according to claim 1, wherein the circular donor DNA further includes a promoter for regulating expression of the donor DNA.

3. The method of claim 2, wherein the promoter is derived from bacteria, yeast, insect, animal, plant or virus.

4. The method of claim 3, wherein the promoter is selected from the group consisting of E. coli lac and trp operons, the tac promoter, the bacteriophage λ

p^L promoter, bacteriophage T7 and SP6 promoters, β

-actin promoter, insulin promoter, human cytomegalovirus (CMV) promoter, HIV-LTR, RSV-LTR, SV40 promoter, baculoviral polyhedrin and p10 promoter.

5. The method of claim 3, wherein the promoter is an inducible promoter.

6. The method of claim 5, wherein the inducible promoter is selected from the group consisting of tetracycline, heat shock, steroid hormone, heavy metal, phorbol ester, adenovirus E1A element, interferon, and serum inducible promoters.

7. The method according to claim 1, wherein the recombination sites on the circular donor DNA and the circular acceptor vector are both lox sites.

8. The method according to claim 1 further comprising steps of transforming, transfecting or transducing a host cell with the recombinant expression vector;

and

selecting the correctly fused recombinant vector based on the selectable phenotype conferred by the selectable marker gene carried by the circular double stranded donor DNA and recombined into the recombinant expression vector.

9. The method according to claim 8 further comprising:

selecting the correctly fused recombinant vector by using a biotin-labeled oligonucleotide which is capable of hybridizing with circular double-stranded donor DNA.

10. The method according to claim 1, wherein the recombinase is selected from the group consisting of the bacteriophage P1 Cre recombinase, yeast FLP recombinase, Inti integrase, bacteriophage λ

, phi 80, P22, P2, 186, and P4 recombinase, Tn3 resolvase, the Hin recombinase, the Cin recombinase, E. coli xerC and xerD recombinases, Bacillus thuringiensis recombinase, TpnI and the β

-lactamase transposons, and the immunoglobulin recombinases.

11. The method of claim 1, the donor or the acceptor recombination site is a DNA sequence recognized by a site-specific recombinase to facilitate site-specific fusion between the circular donor DNA and an acceptor vector containing an acceptor recombination site.

12. The method of claim 1, wherein the donor or the acceptor recombination site is a recombination site recognized by a recombinase, a transposase or an integrase.

13. The method of claim 1, wherein the donor or the acceptor recombination site is a lox site that is recognized by the Cre recombinase of bacteriophage P1.

14. The method of claim 1, wherein the donor or the acceptor recombination site is selected from the group consisting of loxB, loxL, loxR, loxP, loxP3, loxP23, loxΔ

86, loxΔ

117, loxP511, and loxC2.

15. The method of claim 1, wherein the selectable marker is selected from the group consisting of β

-galactosidase, fluorescent protein, secreted form of human placental alkaline phosphatase, β

-glucuronidase, antibiotic resistance genes, yeast seletable markers leu2-d and URA3, apoptosis resistant genes, and antisense oligonucleotides.

16. The method of claim 1, wherein the circular donor DNA further includes an affinity tag.

17. The method of claim 16, wherein the affinity tag is selected from the group consisting of a polyhistidine tract, polyarginine, glutathione-S-transferase, maltose binding protein, a portion of staphylococcal protein A, protein A, and epitope tag.

18. The method of claim 16, wherein the affinity tag is an EE tag.

19. The method of claim 1, wherein the circular donor DNA further includes a polyadenylation signal.

20. The method of claim 1, wherein the circular acceptor vector further comprises a prokaryotic termination sequence selected from the group consisting of the T7 termination sequence, the bacteriophage λ

T_INT, T_L1, T_L2, T_L3, TR₁, TR₂, and T_6S termination signals.

21. The method of claim 1, wherein the circular acceptor vector is a mammalian expression vector.

22. The method of claim 21, wherein the mammalian expression vector contains one or more eukaryotic marker genes, a eukaryotic transcriptional and translational termination signal and a polyadenylation signal.

23. The method of claim 22, wherein the origin of replication functions in bacterial cells.

24. The method of claim 1, wherein the circular acceptor vector is a baculoviral vector.

25. The method of claim 24, wherein the baculoviral vector is a modified Autographa californica multiple nuclear polyhedrosis virus.

26. The method of claim 25, wherein the modified Autographa californica multiple nuclear polyhedrosis virus does not include a polyhedrin promoter.

27. The method of claim 1, wherein the circular donor DNA is generated by ligating a double-stranded linear donor DNA which includes the donor DNA sequence with a double-stranded linear driver DNA which includes the donor recombination site and the at least one selectable marker.

28. The method of claim 27, wherein the linear donor DNA and linear driver DNA contain matching restriction sites.