Bi-directionally cloned random cDNA expression vector libraries, compositions and methods of use
View Patent ↗The present invention provides random cDNA expression vector libraries, comprising expression vectors which comprise random cDNAs positioned in sense and antisense orientation, which are useful for the delivery and expression of a combination of genetic effector types to host cells. Methods for producing these libraries through bi-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.
1. A method for producing a vector, comprising:
a) contacting an mRNA with a primer under conditions to produce a double-stranded cDNA, said primer having a site for a first restriction endonuclease;
b) cleaving said double stranded cDNA to produce cDNA fragments;
c) ligating a double-stranded adaptor to each end of said cDNA fragments to produce adaptor-modified cDNA, said double stranded adaptor having a site for a second restriction endonuclease;
d) contacting said adaptor-modified cDNA with said first restriction endonuclease and said second restriction endonuclease to produce digested cDNA; and,
e) ligating said digested cDNA into a vector.
2. The method of claim 1 , wherein the double stranded adaptor contains a partial restriction site for said first restriction endonuclease, and wherein a dimer of said double stranded adaptors contains a cleavable restriction site for said first restriction endonuclease.
3. The method of claim 1 , wherein said primer is an oligo-dT primer.
4. The method of claim 1 , wherein said primer is a random primer.
5. The method of claim 1 , wherein said first restriction endonuclease is Not1.
6. The method of claim 1 , wherein said second restriction endonuclease is BstX1.
7. The method of claim 1 , wherein said vector is a retroviral vector.