Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1994-01-28
1997-11-11
Fleisher, Mindy
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
4351723, C12P 2102, C12N 1564
Patent
active
056862636
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
This invention relates to recombinant cell culture lines and methods, particularly to the identification and use therein of endogenous nucleic acid sequences for homologous recombination and targeted integration of heterologous nucleic acid sequences. This invention further relates to the therapeutic introduction of nucleic acid sequences into cells or tissue, particularly through targeted integration using homologous recombination; this introduction is useful for gene therapy, or for the creation of transgenic animals.
Gene therapy is the introduction of nucleic acid into a cell or tissue either in vivo or ex vivo. In some instances, the nucleic acid is intended to replace (or act in place of) a functionally deficient endogenous gene, to confer on the host the ability to produce a therapeutic polypeptide, to cause repression of an undesirable gene product, or to stimulate an immune response. Methods are known for introducing nucleic acid into cells in vitro or into such cells or tissues ex vivo, including the insertion of naked DNA or RNA such as by injection into tissue, the use of techniques such as electroporation, the provision of nucleic acid in liposomes or other carrier, the use of a vector such as a virus, retrovirus, phage, plasmid etc., and the reintroduction into a tissue of a cell modified ex vivo to transcribe and express heterologous nucleic acid.
Other researchers have made transgenic animals which transcribe and express heterologous nucleic acid. Such animals have been produced by transfecting germ cells, somatic cells, or embryos with heterologous nucleic acid, suitably implanting the transfected cells and allowing the cells to mature into or stably integrate into adult animals containing the heterologous DNA. A reproducible percentage of such animals transcribe and express the heterologous nucleic acid as protein which can be identified in tissues including blood or serum. Methods for making transgenic animals are described in U.S. Pat. No. 4,396,601.
Recombination involves the opening and cross-wise rejoining of nucleic acid strands within homologous sequences. Intramolecular recombination (recombination between homologous sequences present on a single nucleic acid molecule) has been described in the literature. For example, it has been shown that intramolecular recombination occurs between tandemly arranged, identical, homologous and partially homologous DNA sequences.
Intermolecular recombination is the term used to describe recombination between two different nucleic acid molecules, for example, between two homologous chromosomes during meiosis, or between different viral genomes present in the same infected cell. Intermolecular recombination has been shown in a number of different animal viruses, and this process has been used for the generation of vectors for the cloning and expression of heterologous DNA sequences. Typically, the virus is used as an infectious cloning vector.
It has been shown that, following their introduction into mammalian cells, certain DNA molecules do recombine with each other through shared homologous regions. See, for example, Thomas et al., Cell 44:419-428 (1986), and Zheng et al., Nature 344:170 (1990). Such integration of heterologous DNA into mammalian cells in culture has numerous potential applications, including research, vaccine development, and gene therapy.
Recombination using antisense technology has previously been used to inhibit expression of specific gene products in mammalian cell lines structure of the endogenous target RNA may influence susceptibility to antisense inhibition. Antisense inhibition is currently believed to require an excess of antisense RNA relative to coding mRNA in order to be effective.
Various methods are currently used for the introduction into and amplification of foreign (heterologous) nucleic acid into cultured cells for research purposes as well as for the purpose of overproduction of proteins of interest for pharmaceutical applications. While a lot of knowledge has been accumulated ove
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Fleisher Mindy
Genentech Inc.
Ketter James
Love Richard B.
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