Directionally cloned random cDNA expression vector libraries, compositions and methods of use

• US 6,808,906 B2
• Filed: 05/08/2002
• Issued: 10/26/2004
• Est. Priority Date: 05/08/2002
• Status: Expired due to Term

• Current Assignee: Rigel Pharmaceuticals, Inc.
• Original Assignee: Rigel Pharmaceuticals, Inc.
• Inventors: Yu, Simon, Wu, Xian, Shen, Mary, Payan, Donald
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US10/142,662
• Publication Number:
  US 20030211462A1
• Time in Patent Office: 902 Days
• Field of Search: 435/91.41, 435/320.1, 435/325, 435/252.33, 435/489, 435/91.51
• US Class Current: 435/91.41
• 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
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2740/13043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2800/70 Vectors containing special elements for cloning, e.g. topoisomerase, adaptor sites
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important

Abstract

The present invention provides random cDNA expression vector libraries, comprising expression vectors which comprise random cDNAs positioned in sense orientation. Also provided are random cDNA expression vector libraries, comprising expression vectors which comprise random cDNAs positioned in antisense orientation. Methods for producing these libraries through directional cloning of random cDNAs are also provided. Also provided herein are methods of using these libraries to screen for agents capable of modulating cell phenotype in desirable ways.

Claims

1. A method for producing a vector, comprising:

a) contacting an mRNA with a random primer under conditions suitable for production of a double-stranded cDNA, said random primer having at its 5′

terminus a partial sequence of a restriction site for an infrequently cutting restriction endonuclease;

b) ligating double-stranded adaptors to the ends of said double-stranded cDNA to produce an adaptor-modified cDNA having a complete restriction site sequence for said endonuclease at one end of said double-stranded cDNA;

c) contacting said adaptor-modified cDNA with said restriction endonuclease to produce digested cDNA; and

d) ligating the digested cDNA into a vector.

2. The method of claim 1, wherein said vector is an expression vector.

3. The method of claim 1, wherein said vector comprises a transcriptional regulatory sequence.

4. The method of claim 3, wherein said cDNA is ligated in a sense orientation with respect to said transcriptional regulatory sequence.

5. The method of claim 3, wherein said cDNA is ligated in an anti-sense orientation with respect to said transcriptional regulatory sequence.

6. The method of claim 1, wherein said vector is a retroviral vector.

7. The method of claim 1, wherein said adaptors contain a 3′

overhang ligatable to said vector.

10. A library of vectors made by the method of claim 1.

11. A library of cells containing the library of vectors of claim 10.

12. The method of claim 1, wherein said infrequently cutting restriction endonuclease is Sfi1, BstAP1, PfiM1, Mwo1 or AlwN1.

13. The method of claim 1, wherein said infrequently cutting restriction endonuclease is Sfi1.

8. A vector comprising a cDNA insert flanked on both sides by different restriction sites for an infrequently cutting restriction endonuclease.

9. A cell containing the vector of claim 8.

14. The method of claim 8, wherein said infrequently cutting restriction endonuclease is Sfi1, BstAP1, PfiM1, Mwo1 or AlwN1.

15. The method of claim 8, wherein said infrequently cutting restriction endonuclease is Sfi1.