Genetic tagging strategy for inducing and identifying mutations in a genomic sequence
OwnerClifford Lee Wang, Matthias Wablinventors
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Abstract
The invention features methods and compositions for introducing mutations in an endogenous host cell gene. In one embodiment, the invention features identification of genes that, when mutated, result in production of a phenotype of interest, e.g., tumor formation. In general, the invention provides a random mutagenesis system wherein a non-oncogenic, replicating vector acts as a vehicle to randomly introduce a construct comprising a hypermutation-inducing element into the genome of a host cell. Introduction of the hypermutation element in the host cell genome induces mutations (e.g., point mutations, small deletions, and/or small insertions) in genes adjacent to the integrated hypermutation element.
Claims39
A method for mutating a gene in a host cell genome, the method comprising:
- introducing a hypermutation-inducing construct into a vertebrate host cell, the construct comprising a cis-acting hypermutation element;
- wherein said introducing provides for integration of at least the cis-acting hypermutation element of the construct into a host cell genome and adjacent an endogenous host cell gene so that transcription of the endogenous host gene and the cis-acting hypermutation element facilitates introduction of a mutation into the endogenous host gene to generate a mutated gene.
A method for identifying a proto-oncogene, which gene becomes oncogenic upon introduction of a mutation, the method comprising:
- introducing a hypermutation inducing construct into a non-human animal host, or into any animal cell, including human, the construct comprising a cis-acting hypermutation element, wherein said cis-acting hypermutation element comprises an immunoglobulin intronic enhancer, said introducing providing for integration of at least the cis-acting hypermutation element into a host cell genome and adjacent an endogenous host gene so that transcription of the endogenous host gene and the cis-acting element facilitates production of a mutated host gene having a mutation;
- detecting tumor formation in the host;
- and
- identifying the mutated gene adjacent the cis-acting hypermutation element in nucleic acid of the tumor;
- wherein detection of a tumor having a mutated endogenous gene adjacent the hypermutation element indicates that the endogenous gene is a proto-oncogene.
A method for identification of a gene that becomes oncogenic after introduction of a mutation, the method comprising:
- introducing a hypermutation inducing construct into a murine host, wherein the construct comprises a cis-acting hypermutation element, and wherein said cis-acting hypermutation element comprises a heavy chain large intronic enhancer or a kappa intronic enhancer, said introducing providing for integration of at least the cis-acting hypermutation element into a murine cell genome and adjacent an endogenous gene so that transcription of the endogenous gene and the cis-acting element facilitates production of a mutated gene having a mutation;
- detecting tumor formation in the host;
- and
- identifying the mutated gene adjacent the cis-acting hypermutation element in nucleic acid of the tumor;
- wherein detection of tumors formed as a result of mutation of the endogenous gene indicates that the endogenous gene is a proto-oncogene.
A vertebrate cell having a genomically integrated cis-acting hypermutation element, which element is adjacent and operably linked to a gene endogenous to the vertebrate cell and with which the element is not normally found in nature.
A non-human animal having a genomically integrated cis-acting hypermutation element, which element is adjacent and operably linked to a gene endogenous to the animal and with which the element is not normally found in nature.
A vector comprising a cis-acting hypermutation element, wherein the vector is adapted for integration into a genome of a vertebrate cell, with the proviso that the cis-acting hypermutation element is not operably linked to a gene encoding a reporter polypeptide or a immunoglobulin polypeptide.
Dependencies are inferred from the claim text.