Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1994-05-02
1996-06-18
LeGuyander, John L.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 691, 435 911, 4351723, 4352401, 4352404, 4353201, 536 231, 536 245, C12N 1582, C12N 1511, C12Q 168
Patent
active
055276741
ABSTRACT:
Novel genetic constructs and methods for their use in transforming target cells are disclosed. The genetic constructs, which are particularly adapted for homologous recombination with target-cell genomes, comprise a positively selectable genetic marker and a negative selection system "antagonistic" to the expression of the positively selectable marker. The positively selectable marker is situated in a region of the construct between a first and a second flanking sequence homologous to sequences flanking a desired integration site in the target-cell genome. The negative selection system is situated outside the region. The negative selection system preferably comprises an antisense gene that prevents expression of the positively selectable marker. The positively selectable marker preferably encodes an antibiotic resistance factor. The construct can also include a "gene of interest", also situated between the homologous flanking sequences adjacent the positively selectable marker, for introduction via homologous recombination into the target genome at the desired integration site. Transformed target cells whose genomes homologously recombine with the genetic construct acquire the positively selectable marker and (if present) the gene of interest. Non-homologous recombinants acquire both the positively selectable marker and the negative selection system, thereby losing the ability to survive exposure to the corresponding positive selection agent. Non-recombinants acquire no part of the construct. Thus, homologous recombinants have the exclusive ability to survive exposure to the positive selection agent and can be easily isolated from all other target cells.
REFERENCES:
Tomizawa et al., "Inhibition of ColE1 RNA Primer Formation by a Plasmid-Specified Small RNA," Proc. Natl. Acad. Sci. USA 78:1421-1425 (1981).
Mizuno et al., "Unique Mechanism Regulating Gene Expression: Translational Inhibition by a Complementary RNA Transcript (micRNA)," Proc. Natl. Acad. Sci. USA 81:1966-1970 (1984).
Simons, "Naturally Occurring Antisense RNA Control--A Brief Review," Gene 72:35-44 (1988).
Van der Krol et al., "Modulation of Eukaryotic Gene Expression by Complementary RNA or DNA Sequences," BioTechniques 6:958-976 (1988a).
Van der Krol et al., "Antisense Genes in Plants: An overview," Gene 72:45-50 (1988b).
Izant and Weintraub, "Inhibition of Thymidine Kinase Gene Expression by Anti-Sense RNA: A Molecular Approach to Genetic analysis," Cell 36:1007-1015 (1984).
Smith et al., "Antisense RNA Inhibition of Polygalacturonase Gene Expression in Transgenic Tomatoes," Nature 334:724-726 (1988).
Sheehy et al., "Reduction of Polygalacturonase Activity in Tomato Fruit by Antisense RNA," Proc. Natl. Acad. Sci. USA85:8805-8809 (1988).
Van der Krol et al., "An Anti-Sense Chalcone Synthase Gene in Transgenic Plants Inhibits Flower Pigmentation," Nature 333:866-369 (1988c).
Ecker and Davis, "Inhibition of Gene Expression in Plant Cells by Expression of Antisense RNA," Proc. Natl. Acad. Sci. USA 83:5372-5376 (1986).
Rothstein et al., "Stable and Heritable Inhibition of the Expression of Nopaline Synthase in Tobacco Expressing Antisense RNA," Proc. Natl. Acad. Sci. USA 84:8439-8443 (1987).
Inouye, "Antisense RNA: Its Functions and Applications in Gene Regulation-A Review," Gene 72:25-34 (1988).
Capecchi, "Altering the Genome by Homologous Recomination," Science 244:1288-1292 (1989).
Thomas and Capecchi, "Targeting of Genes to Specific Sites in the Mammalian Genome," Cold Spr. Harb. Symp. Quant. Biol. 51:1101-1113 (1986).
Thomas and Capecchi, "Site-Directed Mutagensis by Gene Targeting in Mouse Embryo-Derived Stem Cells," Cell 51:503-512 (1987).
Smithies et al., "Insertion of DNA Sequences Into the Human Chromosomal .beta.-globin Locus by Homologous Recombination," Nature 317:230-234 (1985).
Hinnen et al., "Transformation of Yeast," Proc. Natl. Acad. Sci. USA 75:1929-1933 (1978).
DeLozanne and Spudich, "Disruption of the Dictyostelium myosin Heavy Chain Gene by homologous Recombination," Science 236:1086-1091 (1987).
Lee et al., "Homologous Recombination and Stable Transfection in the Parasitic Protozoan Trypanosoma brucei," Science 250:1583-1587 (1991).
Horng et al., "Development of a Homologous Transformation System for Aspergillus parasiticus with the Gene Encoding Nitrate Reductase," Mol. Gen. Genet. 224:294-296 (1990).
Gal et al., "Genomic Homologous Recombination in Planta," Embo J. 10:1571-1578 (1991).
Wong and Capecchi, "Homologous Recombination Between Coinjected DNa Sequences Peaks in Early to Mid-S Phase," Molec. Cell. Biol. 7:2294-2295 (1987).
Toulme and Helene, "Antimessenger Oligodeoxyribonucleotides: An Alternative to Antisense RNA for Artificial Regulation of Gene Expression--A Review," Gene 72:51-58 (1988).
Rothstein et al., "Silencing Gene Expression in Plants," In Oxford Surveys of Plant Mol. & Cell Biol. 6:221-246 (1989).
Guerra Daniel J.
Xiang Chengbin
Idaho Research Foundation Inc.
LeGuyander John L.
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