Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-08-10
2001-03-20
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S091410, C435S455000, C435S471000, C435S468000, C800S021000, C800S278000, C514S04400A, C536S023100
Reexamination Certificate
active
06204062
ABSTRACT:
FIELD OF INVENTION
The present invention relates to targeting of genes, and more particularly to targeting of chromosomal gene sequences in eukaryotic cells.
BACKGROUND
Gene targeting by means of homologous DNA recombination is a powerful technique for elucidating the function of eukaryotic genes. Depending upon the precise structure and mode of presentation of the added DNA, targeting can result in complete inactivation of the chromosomal target gene or, alternatively, can alter the target's structure or expression in subtle and well-defined ways (Capecchi, M. R. (1989) Science 244, 1288-1292). A key distinction in gene targeting is whether the vector and approach used are intended to cause gene replacement or gene insertion (Hasty, P. et al. (1991) Mol. Cell. Biol. 11, 4509-4517). In the first case, two homologous recombination events replace the region under study with a selectable gene (FIG.
1
B). In gene insertion, a single recombination event adds the entire foreign DNA molecule to the targeted locus (FIG.
1
C). Gene insertion may be accomplished using either a circular plasmid or one which contains a double strand break within the region of homology, and gene replacement utilizes molecules linearized at a point outside of the homologous regions.
Despite the distinctions drawn between mechanisms of gene replacement and insertion, experiments intended to produce gene replacement often result instead, in a variable proportion of cases, in gene insertion (Hasty, P. et al. (1991) Mol. Cell. Biol. 11, 4509-4517; Thomas, K. R. et al. (1992) Mol. Cell. Biol. 12, 2919-2923; Zhang, H. et al. (1994) Mol. Cell. Biol. 14, 2404-2410). A locus insertionally targeted may, depending upon the vector design, fail to be inactivated; the insertion event is also subject to reversion (Thomas, K. R. et al. (1992) Mol. Cell. Biol. 12, 2919-2923; Hasty, P. et al. (1991) Nature 350, 243-246). Unintended insertional targeting occurs during the transformation of mammalian cells and of the simple eukaryote
Dictyostelium discoideum
(Manstein, D. J. et al. (1989) EMBO J. 8, 923-932; Sun, T. J. et al. (1991) Genes Dev. 5, 572-582). Events of this type are thought to occur via the joining in vivo of the linearized ends of the plasmid, recreating a circular vector capable of single-crossover insertion at the homologous locus. There is evidence of prolific end-to-end joining activity (i.e., DNA ligation) in a wide variety of eukaryotes. Ligation in vivo occurs whether the ends introduced are “sticky,” blunt, or even incompatible (Goedecke, W. et al. (1994) Nucleic Acids Res. 22, 2094-2101; Katz, K. et al. (1990) Ph.D. thesis, University of Massachusetts). It would be desirable to prevent unwanted end-to-end joining of transfecting molecules, and thereby gene insertion, to promote only the desired double cross-over replacement events.
Chang and Wilson (Chang, X.-B. et al. (1987) Proc. Natl. Acad. Sci., USA 84, 4959-4963) observed that 2′3′dideoxy-blocked DNA ends are unable to be ligated in mammalian cells in vivo. There is no information concerning effects of this biochemical procedure on chromosomal integration or gene targeting.
SUMMARY
An embodiment of the invention is a composition of matter for replacing a target sequence in a gene sequence in a eukaryotic cell, comprising: a linear DNA molecule having (i) a desired replacement sequence, and (ii) second and third sequence substantially homologous to non-identical portions of the gene and having proximal and distal ends, the proximal ends flanking the desired replacement sequence; and a terminating nucleotide analog at each end of the DNA molecule. According to this embodiment, the DNA molecule can comprise in addition (iii) a fourth sequence that confers a lethal phenotype and is located at a site selected from the group consisting of the distal ends of each of the second and third sequences, for example, the fourth sequence is a deleterious tRNA gene.
The invention in another embodiment provides also a method of replacing a target sequence in a gene sequence of a eukaryotic cell, comprising: providing a linear DNA molecule having (i) a desired replacement sequence, and (ii) second and third sequences substantially homologous to non-identical portions of the gene and having proximal and distal ends, the proximal ends flanking the desired replacement sequence; adding a terminating nucleotide analog to each end of the DNA molecule; presenting the resulting DNA molecule to the eukaryotic cell such that the resulting DNA molecule enters and transforms the cell; and growing the cell to obtain recombinant progeny cells having the desired replacement sequence inserted into the targeted gene.
In a further embodiment of this method, adding the terminating nucleotide analog to the DNA includes adding a 2′,3′-dideoxynucleotide, for example, adding the 2′,3′-dideoxynucleotide includes using a DNA polymerase. In an alternative embodiment, adding the 2′,3′-dideoxynucleotide includes using terminal deoxynucleotidyl transferase.
According to an embodiment of the method, providing the linear DNA molecule includes digesting a plasmid with a restriction enzyme.
According to another embodiment of the method, the desired sequence confers a selectable phenotype.
According to an embodiment of the method, in providing the DNA molecule, such molecule includes (iii) a fourth sequence that confers a lethal phenotype and is located outside of the sites selected from the group consisting of distal ends of the second and third sequences.
According to an embodiment of the method of providing the DNA molecule with the desired sequence that confers a selectable phenotype, the DNA molecule includes (iii) a fourth sequence that confers a lethal phenotype and is located outside of the sites selected from the group consisting of the distal ends of the second and third sequences.
In another embodiment of the invention, a method is provided in which the targeted replacement of a genomic sequence inactivates a function of the gene. The gene can be in the cell of an animal, and the inactivated function of the gene in a cell of an animal produces an animal model for a human disease. In this embodiment, the human disease can be selected from the group consisting of cystic fibrosis, Lesch-Nyhan syndrome, emphysema, and muscular dystrophy.
In another aspect of this method, the inactivated function of the gene in a cell of an animal eliminates production of an antigen by the animal. In this aspect, the inactivated function of the gene in a cell of an animal produces the antigen causes immune rejection of a tissue or organ donated by the animal.
Another embodiment provided by the invention is a method wherein the gene is in the cell of a plant. According to this embodiment, the inactivated function of the gene in a cell of a plant results in elimination of production of an antigen by the plant, for example, where the antigen in the cell of the plant is a human allergen. In this embodiment, the plant can be a peanut plant,
Arachis hypogaea.
Further, the plant can a Brazil nut plant,
Bertholletia excelsa.
In another aspect of the embodiment provided by this method, the gene is found in the chloroplast DNA in a cell of a plant.
An embodiment of the invention provides a method for correcting a mutation in the gene sequence, for example, a deleterious mutation in the gene sequence, wherein the desired sequence includes a normal allele to a deleterious mutation. The deleterious mutation can be a recessive mutation, for example, a mutation selected from the group consisting of mutations resulting in the diseases: adenosine deaminase deficiency resulting in SCID (severe combined immuno-deficiency), cystic fibrosis, Duchenne muscular dystrophy, phenylketonuria, sickle cell anemia, &agr;-thalassemia, &bgr;-thalassemia, breast cancer, and Crigler-Najjar syndrome.
In another embodiment of the invention, the mutation can be a dominant mutation, for example, a mutation selected from the group consisting of mutations resulting in the diseases: achond
Bromberg & Sunstein LLP
Guzo David
Leffers, Jr. Gerald G.
Trustees of Amherst College
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