Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1997-04-14
2002-04-09
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S069800, C435S325000, C435S358000, C435S366000, C435S235100, C435S455000, C435S456000, C536S023100, C536S023700, C536S024100
Reexamination Certificate
active
06368821
ABSTRACT:
BACKGROUND AND SUMMARY OF THE INVENTION
The ability to exogenously introduce DNA into mammalian cells has become a commonplace and essential part of molecular biology work with many applications. An efficient and reproducible method to accomplish such introduction would be useful, for example, for expressing recombinant proteins, for studying mammalian gene regulation, for functional screening of gene libraries, for creation of transgenic cell lines, and for gene therapy. A number of methods have been used to introduce DNA into mammalian cells.
For example, chemical transfection of cells relies on the treatment of DNA with specific chemicals (e.g., calcium phosphate) or cationic lipids (e.g., lipofectamine). These methods are reasonably reliable although each cell line must be empirically evaluated for optimal efficiency. Also, chemical transfection occasionally suffers from its lack of reproducibility from experiment to experiment and from cell line to cell line. The highest efficiencies are usually achieved with the lipid like compounds. Such compounds, however, often are expensive and many have been demonstrated to be toxic to the cells (1).
A second method to incorporate DNA into mammalian cells is by electroporation. Although this method can be efficient, it is very unreliable and requires a significant amount of handling of the cells. Most cells that become subject to the treatment eventually die during the electric shock, but those that survive have usually taken up the DNA.
A third common technique that exhibits a number of advantages to both transfection and electroporation is infection by virus. Viral infection does not require significant handling of the cells, it is very efficient, high titers of some viruses can be achieved, some viral vector systems have a very large coding capacity, and many cell lines are amenable to this process. However, as attractive as viral systems of gene delivery would appear, significant drawbacks limit or preclude their use. Such drawbacks include cell line specificity, safety concerns, low viral titers, limited vector capacity (in some cases), and cumbersome genomes for genetic manipulations.
The two most commonly used viral vectors are retroviruses and adenoviruses. A primary concern are health and safety issues, particularly for retroviruses. Retroviruses are known causative agents for many types of cancers and infectious diseases. Because their life cycle involves genetic recombination with the host DNA and, because mammalian DNA contain sequences homologous to DNA contained in retroviruses, even vectors that are themselves replication incompetent can be converted to replication competent at a low but measurable frequency. This limits their use in many scientific settings and presents an obstacle to many gene therapy strategies. However, they are utilized in spite of these concerns because they can be propagated at very high titers and libraries can be packaged and infected. Retroviruses only infect actively growing cells and certain cell lines. Another drawback is that the size of foreign DNA that a retroviral vector can incorporate is limited to 5 kb, which may limit its use for many functional genomic cloning strategies.
Adenovirus vectors have a number of advantages over retroviruses. They infect a wide variety of cell lines and, because of their size, can incorporate very large DNA inserts. However, their titers are usually quite low which makes direct library screening difficult to accomplish. In addition, the adenovirus is not benign to either mammalian cells (induction of undesired genes will often occur making certain gene expression studies problematic to evaluate) or people working with the virus, and adverse health effects can result.
Accordingly, there continues to be a need for methods that significantly improve DNA introduction into mammalian cells in any of the areas of efficiency, reproducibility, ubiquity among cell lines and growth phases, and providing for large DNA capacity. It would also be advantageous if one could efficiently introduce primary ligation products (unamplified in another host) so that direct functional cloning is achieved without the need for amplification in
E. coli
, which is inherently biased. In addition, the ability to target specific tissues or cells by using a wholly benign viral system would be favored by many. This would be particularly advantageous for gene therapy.
An object of the present invention is to provide improved systems for gene delivery. According to certain embodiments, such systems will combine the efficiency and high titers of retroviruses, the cell line ubiquity and large size capability of adenovirus, and the benign effect that chemical transfection (by calcium phosphate) has on the cells.
The present invention provides improved DNA delivery systems by transfecting eukaryotic cells with the membrane receptor for a bacteria virus receptor and infecting these cells with the bacteria virus.
According to certain preferred embodiments, the bacterial virus is an
E. coli
virus.
According to certain preferred embodiments, the present inventors have achieved improved gene delivery systems by transfecting a mammalian cell line with the membrane receptor for the
E. coli
bacteriophage lambda and infecting these cells with the
E. coli
virus.
According to certain preferred embodiments, infection involved the Chinese hamster ovary (CHO) bell line. This infection was specific for cells expressing the receptor with the proper membrane targeting signals and stably transfected cell lines resulted.
According to certain preferred embodiments, cells transfected with DNA encoding a membrane receptor for a bacterial virus are provided. Such cells can be used to express DNA contained in bacterial viruses after infection with the virus.
Exploiting this process would have a number of applications including, but not limited to, basic molecular biology studies and gene therapy strategies. Libraries (both genomic and cDNA) could be directly introduced into mammalian cells without the requirement for bacterial amplification, subcloning, or other such manipulations. Large segments of DNA can be introduced (up to 40 kb for cosmids), and direct expression cloning experiments readily can be performed. By controlling the way that the lambda receptor is expressed, one can conceivably control the cell type or the part of the cell cycle that the DNA enters.
REFERENCES:
patent: 5128256 (1992-07-01), Huse et al.
Alting-Mees, M et al., “New Lambda and Phagemid Vectors for Prokaryotic and Eukaryotic Expression,”Strategies in molecular biology, 5:58-61 (1992).
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Chalfie, M. et al., “Green Fluorescent Protein aa a Marker for gene Expression,”Science, 263:802-805 (1994).
Charbit, A. et al., “Permissive Sites and Topology of an Outer Membrane Protein with a Reporter Epitope,”Journal of Bacteriology, 173:262-275 (1991).
Clément, J. and Hofnung, M., “Gene Sequence of the &lgr; Receptor, an Outer membrane proetin ofE. coliK12,”Cell, 27:507-514 (1981).
Gehring, K. et al., “Bacteriophage &lgr; Receptor Site on theEscherichia coliK-12 LamB Protein,”Journal of Bacteriology, 169:2103-2106 (1987).
Greener, A., “E. coliSURE™ Strain: Clone “unclonable” DNA,”Strategies in moleucular biology, 3:5-9 (1990).
Hopp, T. et al., “A Short Polypeptide Marker Sequence Useful for Recombinant Protein Identification and Purification,”Bio/Technology, 6:1204-1210 (1988).
Kaneda, Y., “Virus (Sendai Virus Envelopes)-Mediated Gene Transfer,”Cell Biology: A Laboratory Handbook, pp. 50-57 (Academic Press 1994).
Kuipers, O., “Random Mutagenesis by Using Mixtures of dNTP and dITP in PCR,”Methods in Molecular Biology vol. 57, Chapter 31: In Vitro Mutagenesis Protocols(Humana Press Inc. 1993).
Lappalainen, K. et al., “Comparison of Cell Proliferation and Toxicity Assays Using Two Cationic Liposomes,”Pharmaceutical Research, 11:1127-1131 (1994).
Liu, Z. et al., “A Systematic Comparison of Relat
Chang Hwai Wen
Greener Alan Lewis
Guzo David
Stratagene
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