Cloning system for construction of recombinant expression vectors

• US 6,953,689 B2
• Filed: 02/27/2003
• Issued: 10/11/2005
• 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): Guzo, David
• Assistant Examiner(s): BURKHART, MICHAEL D
• Application Number:
  US10/378,064
• Publication Number:
  US 20040005591A1
• Time in Patent Office: 957 Days
• Field of Search: 435/91.4, 435/91.41, 435/462, 435/320.1, 435/6, 435/5, 435/91.2, 536/23.1
• US Class Current: 435/320.1
• 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

Cloning systems are provided for constructing expression vectors. In one aspect of the invention, a kit is provided for constructing one or more recombinant expression vectors. The kit comprises: a linear driver DNA comprising a promoter sequence, a donor recombination site, and at least one selectable marker, the linear driver DNA being capable of being ligated with one or more linear donor DNA comprising a donor DNA sequence to form one or more circular donor DNA; and a circular acceptor vector comprising an origin of replication and an acceptor recombination site capable of recombining with the circular donor DNA to form the recombinant expression vector for expressing the donor DNA sequence.

Claims

1. A kit for generating a recombinant expression vector for expressing at least one donor DNA sequence, comprising:

a linear driver DNA comprising a promoter sequence, a donor recombination site, and at least one selectable marker, the linear driver DNA not including an origin of replication but being capable of being ligated with one or more linear donor DNA comprising a donor DNA sequence to form one or more circular donor DNA which does not include an origin of replication; and

a circular acceptor vector comprising an origin of replication and an acceptor recombination site capable of recombining with the circular donor DNA to form the recombinant expression vector for expressing the donor DNA sequence.

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

3. The kit according to claim 1, wherein the recombination sites on the linear driver DNA and the circular acceptor vector are both lox sites.

4. The kit according to claim 1, further comprising:

a biotin-labeled oligonucleotide which is capable of hybridizing with the circular donor DNA.

5. The kit according to claim 1, wherein the promoter is from bacteria, yeast, insect, animal, plant or virus.

6. The kit according to claim 5, 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 cytomegalo/virus (CMV) promoter, HIV-LTR, RSV-LTR, SV40 promoter, baculoviral polyhedrin and p10 promoter.

7. The kit according to claim 5, wherein the promoter is an inducible promoter.

8. The kit according to claim 7, 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.

9. The kit according to claim 1, further comprising:

a recombinase capable of recognizing the donor and acceptor recombination sites and catalyzing fusion of the whole sequence of the circular donor DNA into the circular acceptor vector.

10. The kit according to claim 9, 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, Hin recombinase, Cin recombinase, E. coli xerC and xerD recombinases, Bacillus thuringiensis recombinase, TpnI and β

-lactamase transposons, and immunoglobulin recombinases.

11. The kit according to claim 1, wherein the donor or the acceptor recombination site is a lox site that is recognized by the Cre recombinase of bacteriophage P1.

12. The kit according to claim 11, 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.

13. The kit according to 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 selectable markers leu 2-d and URA3, apoptosis resistant genes, and antisense oligonucleotides.

14. The kit according to claim 1, wherein the circular donor DNA further includes an affinity tag.

15. The kit according to claim 14, 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.

16. The kit according to claim 14, wherein the affinity tag is an EE tag.

17. The kit according to claim 1, wherein the circular donor DNA further includes a polyadenylation signal.

18. The kit according to 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.

19. The kit according to claim 1, wherein the circular acceptor vector is a mammalian expression vector.

20. The kit according to claim 19, wherein the mammalian expression vector contains one or more eukaryotic marker genes, a eukaryotic transcriptional and translational termination signal and a polyadenylation signal.

21. The kit according to claim 1, wherein the origin of replication functions in bacterial cells.

22. The kit according to claim 1, wherein the circular acceptor vector is a baculoviral vector.

23. The kit according to claim 22, wherein the baculoviral vector is a modified Autographa californica multiple nuclear polyhedrosis virus.

24. The kit according to claim 23, wherein the modified Autographa californica multiple nuclear polyhedrosis virus does not include a polyhedrin promoter.

25. The kit according to claim 1, wherein the linear donor DNA and linear driver DNA contain matching restriction sites.

26. The kit according to claim 1, wherein the one or more linear donor DNA is a library of DNAs, each comprising a donor DNA sequence independently varying within the library.

27. The kit according to claim 1, wherein the one or more linear donor DNA is generated by PCR amplification of a cDNA library.

28. A kit for generating a recombinant expression vector for expressing at least one donor DNA sequence, comprising:

a linear driver DNA comprising a promoter sequence, a donor recombination site, and at least one selectable marker, the linear driver DNA being capable of being ligated with one or more linear donor DNA comprising a donor DNA sequence to form one or more circular donor DNA; and

a circular acceptor vector that is a mammalian expression vector comprising an origin of replication and an acceptor recombination site capable of recombining with the circular donor DNA to form the recombinant expression vector for expressing the donor DNA sequence.

29. The kit according to claim 28, wherein the mammalian expression vector contains one or more eukaryotic marker genes, a eukaryotic transcriptional and translational termination signal and a polyadenylation signal.

30. A kit for generating a recombinant expression vector for expressing at least one donor DNA sequence, comprising:

a linear driver DNA comprising a promoter sequence, a donor recombination site, and at least one selectable marker, the linear driver DNA being capable of being ligated with one or more linear donor DNA comprising a donor DNA sequence to form one or more circular donor DNA; and

a circular acceptor vector that is a baculoviral expression vector comprising an origin of replication and an acceptor recombination site capable of recombining with the circular donor DNA to form the recombinant expression vector for expressing the donor DNA sequence.

31. The kit according to claim 30, wherein the baculoviral vector is a modified Autographa californica multiple nuclear polyhedrosis virus.

32. The kit according to claim 31, wherein the modified Autographa californica multiple nuclear polyhedrosis virus does not include a polyhedrin promoter.