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
1999-12-23
2003-10-28
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S252300, C435S320100, C435S325000, C435S410000, C536S023500, C530S350000
Reexamination Certificate
active
06638732
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention is in the fields of molecular and cellular biology. More particularly, the invention is directed to mutants of the genes encoding Green Fluorescent Protein (GFP) and the proteins encoded by these mutants. The mutant GFPs are used to allow detection of eukaryotic and prokaryotic cells transfected or transformed with extrinsic genes, and to label proteins of interest to facilitate their localization within viable cells.
2. Related Art
Transfection of Foreign Genes
To study the function of a gene, a technique that is commonly employed is the transfer of the gene into a new cellular environment. This process, called “transfection,” provides several advantages to the genetic scientist. For example, the cellular protein encoded by the gene can often be more easily studied by transferring the gene into a cell or organism that normally does not produce the protein, and then examining the effect of this protein on the host cell. The existence and function of regulatory genetic sequences (e.g., promoters, inhibitors and enhancers) may be elucidated by transfection of foreign genes into cells containing the regulatory sequences. The transfer of non-native or altered genes into a host cell also allows for large-scale production of the proteins encoded by the genes, a process upon which much of the current biotechnology industry is based. Transfection of plant embryos with foreign genes has provided genetically engineered plants that are more resistant to adverse environmental conditions or that are more nutritionally rich. Finally, gene transfer methods allow the introduction of new or mutated genes into whole organisms. This latter capability provides the opportunity for the construction of stable models of mammalian diseases, for large-scale production of proteins in the milk of transgenic lactating animals, and for the possibility of genetic therapy for certain diseases.
A variety of techniques has been used to transfect non-native genes into cells (reviewed in Sambrook, J., et al.,
Molecular Cloning, a Laboratory Manual
, 2nd Ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, pp. 16.30-16.55 (1989); Watson, J. D., et al.,
Recombinant DNA
, 2nd Ed, New York: W. H. Freeman and Co., pp. 213-234 (1992)). These techniques include biological methods such as the use of viruses (e.g., adenovirus or certain retroviruses for mammalian cells, baculovirus for insect cells and bacteriophages for bacterial cells) or bacteria (e.g., Agrobacterium for plant cells), chemical methods such as calcium phosphate precipitation, DEAE-dextran-mediated endocytosis or liposome-mediated transfection, and physical methods such as electroporation or direct microinjection. For transfection of mammalian cells, the techniques most commonly employed currently are virus-mediated transfection, lipofection and electroporation.
Detection of Gene Transfer
Regardless of the method used, however, simply attempting to transfect a cell does not guarantee that a majority (or even any) of the target cells will take up and/or express the exogenous DNA. Indeed, it has been suggested that the success rate of even the most optimal techniques used for transfection results in stable transfer of exogenous DNA is far less than 1% (Watson, J. D., et al.,
Recombinant DNA,
2nd Ed., New York: W. H. Freeman and Co., pp. 216, 218 (1992)). Thus, it is usually critical to determine which target cells have received and/or incorporated the gene(s) being transfected, for which a number of methodologies have been used.
Expression
The most obvious of these methods is to simply examine the target cells for expression of the exogenous gene. In this method, the transfected cells are grown in vitro and assayed for the presence of the protein encoded by the transferred gene. These assays are usually accomplished using immunological techniques such as Western blotting, ELISA or RIA. This type of technique is only useful, however, if the protein is produced in relatively high amounts (generally at the microgram level or above) and if suitable antibodies are available, neither of which is the case for some transfected genes.
In those cases where protein expression cannot be examined, incorporation of exogenous genes can be determined by assaying the target cells for production of the mRNAs corresponding to the transferred genes. One very common technique for this determination is Northern blotting (Alwine, J. C., et al.,
Proc. Natl. Acad Sci. USA
74:5350-5354, 1977), in which RNA molecules are isolated from cells, separated by gel electrophoresis and electroblotted onto a solid support (e.g., nitrocellulose or nylon). The solid support is then overlaid with radiolabelled cDNAs corresponding to the transfected gene, which hybridize on the solid support to their complementary mRNAs. After exposing the blot to photographic film, the samples containing the expressed transgene are easily determined. While this method is more sensitive than those directly measuring protein expression, Northern blotting still relies on actual expression of the gene by the target cells, which is not always the case.
Selection
Another method for determining gene transfer, alternative to directly measuring gene expression, is to examine the effect of the gene on the transfected cells. For example, some transfected genes will confer upon their host cells the ability to grow in selective culture media or under some other environmental stress which non-transfected cells cannot tolerate. Genes of interest are often engineered into sequences conferring, for example, antibiotic resistance upon the recipient cells. Transfectants with these constructs will thus carry not only the gene of interest but also the antibiotic resistance gene which allows them to grow in antibiotic-containing media. Since non-transfected cells will not possess this resistance, any cell able to grow in media containing antibiotic will contain the resistance marker (the so-called “selectable marker”) and the transgene that is linked to it. Selectable markers commonly used in such an approach are the neomycin (neo), ampicillin (amp) and hygromycin (hyg) resistance genes.
In the same way, selectable markers conferring on the transfected cells a metabolic advantage (e.g., ability to grow in nutrient-deficient media) have been used successfully. Examples of these types of selectable markers include thymidine kinase (Bacchetti, S., and Graham, F. L.,
Proc. Natl. Acad. Sci. USA
74:1590-1594 (1977); Wigler, M., et al.,
Cell
11:223-232 (1977)) and xanthine-guanine phosphoribosyltransferase (Mulligan, R. C., and Berg, P.,
Proc. Natl. Acad Sci. USA
78:2072-2076 (1981)), which impart to their recipients the ability to grow, using metabolic rescue pathways encoded by the marker genes, in media that inhibit vital metabolic pathways in non-transfected cells. Again, any cells able to grow in such media will contain the transgene linked to the marker gene.
Selection methods such as these often require weeks of culturing of the cells, continuously under selective pressure, to provide a relatively pure population of stable transfectants. Many uses of transfected cells, however, are conducted within hours of transfection, far too soon to determine transfection success using either the expression or selection methods described above. These types of applications are facilitated by a third approach—the use of “reporter genes”.
Reporter Genes
Reporter genes are analogous to selectable markers in that they are co-transfected into recipient cells with the gene of interest, and provide a means by which transfection success may be determined. Unlike selectable markers, however, reporter genes typically do not confer any particular advantage to the recipient cell. Instead reporter genes, as their name implies, indicate to the observer (via some phenotypic activity) which cells have incorporated the reporter gene and thus the gene of interest to which it is linked. A number of reporter genes have been used, including those operating by biochemical
Invitrogen Corporation
Slobodyansky Elizabeth
Sterne Kessler Goldstein & Fox P.L.L.C.
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