Process for gene targeting and genome manipulations

Details Process for gene targeting and genome manipulations Process for gene targeting and genome manipulations

1994-12-12

1998-08-11

Chambers, Jasemine C.

Chemistry: molecular biology and microbiology

Treatment of micro-organisms or enzymes with electrical or...

Modification of viruses

4351721, C12N 1509, C12N 1563, C12N 1587

Patent

active

057926336

DESCRIPTION:

BRIEF SUMMARY
TECHNICAL FIELD

A process to detect illegitimate recombination and restriction enzyme mediated recombination in the yeast Saccharomyces cerevisiae, in the slime mold Dictyostelium discoideum and in mammalian cells.


BACKGROUND ART

More than 3,000 diseases are caused by mutations such as, for example, hemophilia, Tay-Sachs disease, Duchenne's muscular dystrophy, Huntington's disease, alpha-thalassemia, Lesch Nyhan syndrome, etc. Most of these diseases cannot be treated medically.
The science of gene therapy is in its infancy. Gene therapy aims to cause a reversion of the genetic basis of a disease and, thus after one successful treatment, to cure the patient for life; see, e.g., pages 411-441 of a book edited by R. Kucherlapati entitled "Gene Transfer" (Plenum Press, New York, 1986).
Gene therapy attempts to determine the existence and location of a mutated gene and, thereafter, to add a wild type copy of the mutated gene to the cells to thereby replace the mutated gene with the wild type copy; this procedure is often referred to as "gene targeting."
In mammalian cells, however, it is known that genes introduced into such cells integrate into the DNA of the cell primarily at nonhomologous sites; thus, instead of replacing a mutated gene, the wild type copy will be introduced at another locus in the DNA. The DNA so integrated will be more likely to cause mutations than it would have been prior to such integration, for it is known that random integration of genes into mammalian cells is mutagenic. See, e.g., W. King et al., "Insertion mutagenesis of embryonal carcinoma cells by retroviruses," Science 228:554-558, 1985. The random integration of genes into the DNA of mammalian cells, in addition to causing mutations, also causes problems in the expression of the wild type gene. As is known to those skilled in the art, wild type genes situated at their proper loci are usually expressed only in a pattern specific for that particular gene. Thus, for example, certain genes are expressed in an animal's liver and not in its brain. However, when wild type genes are randomly integrated into the DNA of mammalian cells, they are generally not expressed at all and, in those cases where they are expressed, they are usually not expressed in the proper pattern.
Furthermore, in addition to causing mutations and improper expression or no expression, the random integration of wild type genes into the DNA of mammalian cells will not remove the disease-causing gene from the cell. Since such a disease-causing gene sometimes is dominant, the random integration of the wild type genes into such DNA is at best ineffective. There has been a long-felt need for a process for specifically removing the disease-causing gene from the DNA of a cell and replacing it with a wild type gene at a sufficiently high frequency so that the diseased cell will be cured. The slime mold Dictyostelium discoideum is a model system for developmental genetics since for part of its life cycle it is a unicellular amoebae and as such it is very well amenable to molecular biology approaches.
However, one of the most important problems with the development of Dictyostelium as research tool is that cloning by functional complementation with plasmid-borne genomic libraries has not been successful for developmental genes where direct selection cannot be applied. Transposon tagging has been successfully used in other organisms such as Drosophila and Caenorhabditis for the isolation of developmental genes. However, because of the lack of the ability to mobilize transposable elements, this strategy is not possible in Dictyostelium. A method for insertional mutagenesis would be a breakthrough for developmental biology with Dictyostelium and many other organisms.
About fifteen years ago some experiments were reported which allegedly, with the use of a restriction enzyme, incorporated a certain gene fragment into a bacterial plasmid. In an article by Shing Chang and Stanley N. Cohen entitled "In vivo site-specific genetic recombination promoted by the EcoRI restriction

REFERENCES:
Gusew et al., "Linear DNA must have free ends to transform rat cells efficiently". Mol. Gen. Genet. 206: 121-125. 1987.
Kuspa et al., "Tagging developmental genes in Dictyostelium by restriction enzyme-mediated integration of plasmid DNA", Proc. Natl. Acad. Sci. USA 89: 8803-8807. Sep. 15, 1992.
Schiestl et al. "Integration of DNA fragments by illegitimate recombination in Saccharomyces cerevisiae", Proc. Natl. Acad. Sci. USA 88: 7585-7589. Sep. 1, 1991.
Marx, "Dictyostelium researchers expect gene bonanza", Science 258: 402-403. Oct. 16, 1992.
Grimm et al. "Observations on integrative transformation in Schizosaccharomyces pombe", Mol. Gen. Genet. 215: 87-93. 1988.
Chang et al. "In vivo site-specific genetic recombination promoted by the EcoRI restriction endonuclease" Proc. Natl. Acad. Sci. USA 74: 4811-4815. Nov. 1977.

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