Bi-directionally cloned random cDNA expression vector libraries, compositions and methods of use
AssigneeRigel Pharmaceuticals, Inc.
InventorsJames Lorens, Jakob M. Bogenberger
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Abstract
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.
Claims20
An expression vector library, comprising a plurality of expression vectors, each comprising:
- i) a first nucleic acid comprising a cDNA;
- and
- ii) a second nucleic acid which encodes a detectable protein;
- iii) a transcriptional regulatory sequence recognized by a host cell transduced with the expression vector;
- and
- iv) a translational regulatory sequence recognized by a host cell transduced with the expression vector;
- wherein said first and said second nucleic acids form a fusion nucleic acid, wherein said fusion nucleic acid is operably linked to the transcriptional regulatory sequence, and wherein the plurality of expression vectors taken together comprises:
- i) at least a first expression vector comprising a cDNA operably linked to said transcriptional regulatory sequence in antisense orientation;
- ii) at least a second expression vector comprising a cDNA operably linked to said transcriptional regulatory sequence in sense orientation and operably linked to said translational regulatory sequence such that said cDNA is translated in the same frame as the corresponding mRNA;
- and
- iii) at least a third expression vector comprising a cDNA operably linked to said transcriptional regulatory sequence in sense orientation and operably linked to said translational regulatory sequence such that said cDNA is translated out of frame with respect to the corresponding mRNA.
3 The expression vector library according to claim 2, wherein said cDNA is a cDNA restriction fragment.
A method of screening for a bioactive agent capable of altering a cellular phenotype, comprising:
- a) providing a cellular library comprising a retroviral expression vector library comprising a plurality of retroviral expression vectors, each comprising:
- i) a first nucleic acid comprising a cDNA fragment;
- ii) a second nucleic acid which encodes a detectable protein;
- iii) a transcriptional regulatory sequence recognized by a host cell transduced with the expression vector;
- iv) a translational regulatory sequence recognized by a host cell transduced with the expression vector;
- wherein said first and said second nucleic acids form a fusion nucleic acid, wherein
- said fusion nucleic acid is operably linked to the transcriptional regulatory region, and
- wherein the plurality of retroviral expression vectors taken together comprises:
- i) at least a first retroviral expression vector comprising a cDNA operably linked to said transcriptional regulatory sequence in antisense orientation;
- ii) at least a second retroviral expression vector comprising a cDNA operably linked to said transcriptional regulatory sequence in sense orientation and operably linked to said translational regulatory sequence such that said cDNA is translated in the same frame as the corresponding mRNA;
- and
- iii) at least a third retroviral expression vector comprising a cDNA operably linked to said transcriptional regulatory sequence in sense orientation and operably linked to said translational regulatory sequence such that said cDNA is translated out of frame with respect to the corresponding mRNA;
- and
- b) screening said cellular library for a cell with an altered phenotype.
Dependencies are inferred from the claim text.