Multicellular living organisms and unmodified parts thereof and – Method of making a transgenic nonhuman animal
Reexamination Certificate
1998-06-24
2004-12-28
Woitach, Joe (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of making a transgenic nonhuman animal
C800S008000, C800S014000, C800S018000, C800S020000, C800S022000, C435S441000, C435S442000, C435S443000, C435S444000, C435S445000, C435S446000, C435S447000, C435S448000, C435S455000, C435S462000, C435S463000, C435S325000
Reexamination Certificate
active
06835867
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods of producing modifications in genes of interest in a cell. In particular, the invention provides methods for using nucleic acid sequence-modifying agents to introduce modifications in any gene of interest in the genome of a cell. Also provided are sets of cells which contain at least one modification in any gene of interest. The methods and compositions of the invention are useful in determining the function of the gene of interest.
BACKGROUND OF THE INVENTION
With the completion of the Human Genome Program approaching, there is an increasing interest in studying the function of genes, particularly those involved in human development and disease. While mapping and nucleotide sequencing of genes is an important first step for understanding the function of genes, the physical characterization of the structure of a gene does not provide insight into the function of that gene in the context of a multicellular organism.
For example, prior art approaches to determining gene function in mammals have relied on targeting mutations to specific genes in embryonic stem (ES) cells, or on genome-wide mutagenesis techniques designed to mutate all genes of an organism (e.g., mice). For example, “knock-out” mutations in ES cells have been widely used to target mutations to specific genes. “Knock-out” mutations shut off or alter gene expression and are currently used to produce a phenotype in the whole animal which reflects the function of the knocked-out gene. This approach has identified many genes which are associated with cancer and other human genetic diseases, and relies either on phenotype-based screens (i.e., screening for a particular phenotype) or on gene-based screens (i.e., screening for a particular alteration in the genome). Phenotype-based screens have primarily been conducted using mice, and involve characterization of thousands of mutagenized mice for specific diseases and traits [Russell et al., Proc. Natl. Acad. Sci. USA 76:5818-5819, 1979; Hitotsumachi et al., Proc. Natl. Acad. Sci. USA 82:6619-6621; Shedlovsky et al., Genetics 134:1205-1210; Marker et al., Genetics 145:435-443, 1997]. While the phenotype-based approach has the advantage that no assumption is made with respect to which genes are associated with a given disease or disorder, it is nevertheless very costly when using organisms such as mice since it requires the maintenance of several lines of mutagenized whole organisms. Furthermore, it is unclear whether phenotype-based screens permit conducting saturation screens for both dominant and recessive mutations of all mouse genes.
Gene-based screens have been carried out in whole animals and in embryonic stem (ES) cells. This approach involves identifying the organism's genes or the ES cell genes which have been mutated. Homologous recombination and retroviral insertion are commonly used in ES cells [Zambrowicz et al. (1998) Nature 392:608-611]. Although mutagenesis by homologous recombination is becoming routine, it remains cumbersome and expensive. Similarly, while the genome-wide approach to mutagenizing ES cells by retroviral insertional mutagenesis allows the generation of a large number of mutagenized ES cells in a cost effective manner, this approach produces only one, or a limited number of, alleles of a given gene. Additionally, the class of mutations that can be produced with this approach is limited to those mutations which result from integration of a retroviral element. Thus, mutations caused by, for example, single amino acid changes in the protein cannot be produced using this approach. In many instances, for example, it may be desirable to generate mutations which cause single amino acid changes that merely modify gene function (e.g., by generating hypomorphic alleles that express the gene with a reduced efficiency) or that give rise to a new trait in the animal (e.g., by generating dominant neomorphic alleles which result in a gain of function). The generation of hypomorphic and neomorphic alleles of a gene in a model organism by single amino acid substitutions may be desirable to create a model organism for a human trait or disease in which gene function is modified rather than destroyed.
Accordingly, what is needed are methods for determining gene function which may efficiently be applied on a genome-wide scale, which generate more than one mutation in a gene of interest, and which do not only abrogate the function of the gene.
SUMMARY OF THE INVENTION
The invention provides methods for generating an allelic series of modifications in any gene of interest contained in a cell using nucleic acid sequence-modifying agents. In particular, the invention provides a method of producing a modification in a gene of interest contained in a cell, comprising: a) providing: i) a plurality of target cells capable of being cultured; ii) an agent capable of producing at least one modification in the gene of interest in the target cell; b) treating the target cells with the agent under conditions such that a mixture of cells is produced, the mixture of cells comprising cells having an unmodified gene of interest and cells having a is modified gene of interest; and c) isolating the cells having a modified gene of interest.
In one preferred embodiment, the methods of the invention further comprise step d) comparing the nucleotide sequence of the gene of interest in the cells having a modified gene of interest with the nucleotide sequence of the gene of interest in the cells having an unmodified gene of interest. In a more preferred embodiment, the methods further comprise e) manipulating the cells having a modified gene of interest to generate an organism comprising the modification in the gene of interest. In an alternative more preferred embodiment, the method further comprises prior to step d) amplifying the modified gene of interest to produce an amplified modified gene of interest. In yet a more preferred embodiment, the method further comprises prior to step d) sequencing the amplified modified gene of interest.
Without intending to limit the methods of the invention to any particular modification, in one embodiment, the modification is selected from the group consisting of mutation, mismatch, and strand break. In a preferred embodiment, the mutation is selected from the group consisting of deletion, insertion and substitution. In another preferred embodiment, the strand break is selected from the group consisting of single-strand break and double-strand break. While it is not intended that the scope of the invention be limited to any particular type or source of target cell, in one embodiment, the target cell is derived from an organism selected from the group consisting of non-human animal, plant, protist, fungus, bacterium, and virus. In a preferred embodiment, the non-human animal is a mammal. In a more preferred embodiment, the mammal is a mouse. In an alternative preferred embodiment the non-human animal is zebrafish. In another embodiment, the target cell is an embryonic stem cell.
The invention is not intended to be limited to any particular type or class of agent capable of producing at least one modification in the gene of interest. However, in one preferred embodiment, the agent is selected from the group consisting of N-ethyl-N-nitrosurea, methylnitrosourea, procarbazine hydrochloride, triethylene melamine, acrylamide monomer, chlorambucil, melphalan, cyclophosphamide, diethyl sulfate, ethyl methane sulfonate, methyl methane sulfonate, 6-mercaptopurine, mitomycin-C, procarbazine, N-methyl-N′-nitro-N-nitrosoguanidine,
3
H
2
O, urethane, ultraviolet light, X-ray radiation, and gamma-radiation. The invention further provides a method of producing an allelic series of modification in a gene of interest contained in a cell, comprising: a) providing: i) a plurality of target cells capable of being cultured; ii) an agent capable of producing at least one modification in the gene of interest in the target cell; b) treating the target cells with the agent under
Avner Ellis D.
Magnuson Terry R.
Thomas James W.
Woychik Richard P.
Medlen & Carroll LLP
Woitach Joe
LandOfFree
Allelic series of genomic modifications in cells does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Allelic series of genomic modifications in cells, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Allelic series of genomic modifications in cells will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3283049