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US 6,830,892 B2Granted
Lentiviral vector system for high quantity screening
AssigneeDana-Farber Cancer Institute, Inc.
InventorsWayne A. Marasco, Sandra Ogueta
Life of this patent
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Abstract
A method of screening for a target molecule from a group of potential target molecules is described. This method involves using a library of lentiviral vectors where the members encode the group of target molecules, then transducing a group of cells and screening the transduced cell for a desired phenotype. The cell(s) displaying the desired phenotype is selected and the target molecule is identified.
Claims16
A method of screening for a target molecule from a group of target molecules comprising:
- (a) transducing a plurality of cells with a plurality of lentiviral virions, wherein the lentiviral virions are produced by:
- co-transfecting a producer cell with at least (1) one vector containing a lentiviral gag gene encoding a lentiviral gag protein, wherein the lentiviral gag gene is operably linked to a promoter and a polyadenylation sequence, (2) a second vector containing an env gene encoding a functional envelope protein, wherein the env gene is operably linked to a promoter and a polyadenylation sequence;
- and (3) a lentiviral pol gene encoding a lentiviral pol protein on one of the first two vectors or on at least a third vector, wherein said lentiviral pol gene is operably linked to a promoter and a polyadenylation sequence;
- wherein said vectors do not contain sufficient nucleotides to encode the lentiviral gag and pol and the envelope protein on a single vector;
- and
- wherein said vectors do not contain nucleotides of a lentiviral genome referred to as a packaging segment to effectively package lentiviral RNA;
- and wherein the lentiviral proteins and the envelope protein when expressed in combination form a lentivirus virion containing an envelope protein around a lentiviral capsid;
- and (4) a packaging vector containing a nucleic acid sequence encoding a target molecule selected from a plurality of target molecules, wherein the nucleic acid sequence is operably linked to a promoter and a lentiviral packaging sequence including the portion of the lentiviral long terminal repeat (LTR) sequences necessary to package the lentiviral RNA into the lentiviral virion;
- wherein the virion further contains a marker gene;
- (b) identifying transduced cells by screening for the presence of a marker expressed by the marker gene;
- (c) screening for a cell displaying a desired phenotype;
- and
- (d) identifying the target molecule present in the cell displaying the desired phenotype.
10 more elements
Dependencies are inferred from the claim text.