Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-05-08
2004-02-17
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S004000, C435S007100, C435S021000
Reexamination Certificate
active
06692913
ABSTRACT:
FIELD OF INVENTION
The invention relates to a method for the parallel determination of cell vitality and efficiency using a reporter gene after gene transfer in eukaryotic cells in the same culture vessel, as well as to a dual test system.
BACKGROUND
The application of DNA in eukaryotic cells is an important technique in molecular biology. For this purpose, a series of methods were described, such as the calcium phosphate precipitation method (Graham-F L; Eb-AJ-van-der (1973), a new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52: 456-67), various virus-mediated methods (Rosen-C A; Sodroski-J G; Haseltine-W A (1985). The location of cis-acting regulatory sequences in the human T cell lymphotropic virus type III (HTVL-III/LAV) long terminal repeat, Cell 41: 813-23), the application of DNA with the help of cationic liposomes (Felgner-P L; Gadek-T R; Holm-M; Roman-R; Chan-H W; Wenz-M; Northrop-J P; Ringoldi-G M; Danielson-M (1987), Lipofection: a highly efficient, lipid-mediated DNA transfection procedure, Proc.-Natl-Acad-Sci-USA, 84: 7413-7) or of polymers (Fauci-A S (1986), Current issues in developing a strategy for dealing with the acquired immunodeficiency syndrome, Proc-Natl-Acad-Sci-USA, 83: 9278-83; Mosca-J D; Bednarik-D P; Raj-N B; Rosen-C A; Sodroski-J G; Haseltine-W A; Pitha-P M (1987), Herpes simplex virus type 1 can reactivate transcription of latent human immunodeficiency virus, Nature, 325: 67-70: Gendelman-HE; Phelps-W; Feigenbaum-L; Ostrove-J M; Adachi-A; Howley-P M; Khoury-G; Ginsberg-H S; Martin-M A (1986), Transactivation of the human immunodeficiency virus long terminal repeat sequence by DNA viruses, Proc.-Natl-Acad-Sci-USA, 83: 9759-63 and with the help of physical methods (O'Hare-P; Hayward-G S (1985), Evidence for a direct role for both the 175,000 and the 110,000 molecular weight immediate-early proteins of herpes simplex virus in the transactivation of delayed-early promoters, J-Virol. 53: 751-60, Gorman-C M; Moffat-L F; Howard-B H (1982), Recombinant genomes which express chloramphenicol acetyl transferase in mammalian cells, Mol-Cell-Biol. 2: 1044-51).
A series of so-called reporter genes is used, such as the LacZ gene (Lim-K); Chae-C B (1989), to investigate the efficiency of the gene application in the case of in vitro applications. A simple assay for DNA transfection by incubation of the cells in culture dishes with substrates for &bgr;-galactosidase is described in Biotechniques, 7: 576-9), the luciferase gene (Nordeen-S K (1988), Luciferase reporter gene vectors for analysis of promoters and enhancers, Biotechniques, 6: 454-8), the chloramphenicol acetyl transferase gene (Gorman et al. (1982), see above) and others. In comparison to other reporter genes, the LacZ gene offers various advantages. First of all, it is possible to stain individual cells, the transfected cells, expressing the reporter gene, with the help of so-called X-Gal staining (Lojda-Z; Slaby-J; Kraml-J; Kolinska-J (1973), Synthetic substrates in the histochemical demonstration of intestinal disaccharidases, Histochemie, 341-9). Furthermore, the total gene expression of a larger number of cells can be determined with the help of a simple color test (Lim and Chase 1989, see above). In contrast to a large number of other reporter genes, these determinations of the gene expression can be carried out in any laboratory, since only the measurement of the color developed is required, but no radioactive or luminometeric measurements.
Since most techniques, which apply DNA in eukaryotic cells, are accompanied by some cell damage, the determination of cell vitality after a gene transfer or the determination of the toxicity is of importance for characterizing the efficiency and safety of the method. A series of methods are available for determining the toxicity of compounds, which generally involve the measurement of the activity of a representative enzyme of the cells or the manual counting of the living cells. The enzymatic methods have gained the widest acceptance for investigating a large number of samples. In this connection, especially the so-called MTT test (Mossman-T (1983): Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J-Immunol-Methods, 65: 56-63) as well as the acid phosphatase test (Connolly-D T, Knight-M B, Harakas-N K, Witwer-A J, Feder-J (1986), Determination of the number of endothelial cells in culture using an acid phosphatase assay, Anal-Biochem. 152: 136-40), for which the activity of mytochondrial dehydrogenase (MTT) or of cytosolic acid phosphatases is determined are of particular importance here. Until now, there are only methods which determine, in separate batches either the efficiency of the method by way of the reporter gene activity or methods which determine the toxicity of the method by way of an enzyme activity which is representative of the number of cells.
DESCRIPTION OF THE INVENTION
The invention surprisingly enables a combination of a modified acid phosphatase assay with a colorimetric or fluorimetric reporter gene assay for the simultaneous determination of the cell vitality and of the expression of the reporter gene assay, suitably a &bgr;-galactosidase assay for the simultaneous determination of the cell vitality and of the expression of the reporter gene.
The present invention is a process for determining cell vitality and efficiency after gene transfer in eukaryotic cells, which comprises determining in a reaction vessel the cell count of the transfected cells by a representative enzyme activity, and ascertaining in the same reaction vessel the efficiency of the gene transfer by determining the reporter gene activity after changing the ionic strength and pH.
The present invention is also a dual test system for the above process, which comprises (a) a vitality assay with a substrate for determining acid phosphatase activity, and a buffer for cell lysis, and (b) a reporter gene assay with a substrate for the reporter assay, and a buffer for determining the efficiency.
Groth Detlev
Reszka Regina
Davis Katherine F
Goodwin & Procter LLP
Guzo David
Max-Delbruck-Centrum fur Mdekular Medizin
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