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US 7,029,848 B2Granted
High throughput screening of gene function using libraries for functional genomics applications
AssigneeGalapagos Genomics N.V.
InventorsRonald Vogels, Abraham Bout, Helmuth van Es, Govert Schouten
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Abstract
Novel means and methods for their use are provided to determine the function of the product(s) of one or more sample nucleic acids. The sample nucleic acids are synthetic oligonucleotides, DNA, or cDNA and encode polypeptides, antisense nucleic acids, or GSEs. The sample nucleic acids are expressed in a host by a vehicle to alter at least one phenotype of the host. The altered phenotype(s) is/are identified as a means to assign a biological function to the product(s) encoded by the sample nucleic acid(s).
Claims56
A method for determining the function of a unique nucleic acid present in a library, said method comprising:
- (a) providing a library of a multitude of unique expressible nucleic acids, said library including a multiplicity of compartments, each of said compartments consisting essentially of one or more adenoviral vector comprising at least one unique nucleic acid of said library in an aqueous medium, wherein said adenoviral vector is capable of introducing said nucleic acid into a host cell, is capable of expressing the product of said nucleic acid in said host cell, and is deleted in a portion of the adenoviral genome necessary for replication thereof in said host cell;
- (b) transducing a multiplicity of host cells with at least one adenoviral vector comprising at least one unique nucleic acid from said library;
- (c) incubating said host cells to allow expression of the product of said nucleic acid;
- and
- (d) determining the function of said nucleic acid.
2 more elements
A method for determining the function of a unique nucleic acid present in a library, said method comprising:
- (a) growing a plurality of cell cultures containing at least one cell, said one cell expressing adenoviral sequence consisting essentially of E1-region sequences and expressing one or more functional gene products encoded by at least one adenoviral region selected from an E2A region and an E4 region;
- and
- (b) transfecting, under conditions whereby said recombinant adenovirus vector library is produced, said at least one cell in each of said plurality of cell cultures with
- i) an adapter plasmid comprising adenoviral sequence coding, in operable configuration, for a functional Inverted Terminal Repeat, a functional encapsidation signal, and sequences sufficient to allow for homologous recombination with a first recombinant nucleic acid, and not coding for E1 region sequences which overlap with E1 region sequences in said at least one cell, for E1 region sequences which overlap with E1 region sequences in a first recombinant nucleic acid, for E2B region sequences other than essential E2B sequences, for E2A region sequences, for E3 region sequences and for E4 region sequences, and further comprises a unique nucleic acid sequence and promoter operatively linked to said unique nucleic acid sequence;
- and
- ii) a first recombinant nucleic acid comprising adenoviral sequence coding, in operable configuration, for a functional adenoviral Inverted Terminal Repeat and for sequences sufficient for replication in said at least one cell, but not comprising adenoviral E1 region sequences which overlap with E1 sequences in said at least one cell, and not comprising E2A region sequences or E4 region sequences expressed in said plurality of cells which would otherwise lead to production of replication competent adenovirus wherein said first recombinant nucleic acid has sufficient overlap with said adapter plasmid to provide for homologous recombination resulting in production of recombinant adenoviral vectors in said at least one cell;
- (c) incubating said plurality of cells under conditions which result in the lysis of said plurality of cells facilitating the release of said recombinant adenoviral vectors containing said unique nucleic acid;
- and
- (d) transferring an aliquot of said adenoviral vectors into a corresponding plurality of host cell cultures consisting of cells in which said vectors do not replicate, but in which said nucleic acids are expressible;
- (e) incubating said host cells to allow expression of the product of said nucleic acid;
- and
- (f) observing said host cell for changes in said host cell.
9 more elements
Dependencies are inferred from the claim text.