Cells, genetically modified with optical marker

Details Cells, genetically modified with optical marker Cells, genetically modified with optical marker

1998-06-16

2003-01-21

Brusca, John S. (Department: 1631)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Using tissue cell culture to make a protein or polypeptide

C435S069100, C435S455000, C436S072000

Reexamination Certificate

active

06509174

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention concerns a method for obtaining mammalian cells, genetically modified (stably transfected) with a fluorescent marker, preferably green fluorescent protein (GFP), as well as mammalian cells with especially high fluorescence.
The use of fluorescent proteins as reporter molecules for gene expression is already known. One such reporter molecules is the green fluorescent protein (GFP). Said marker protein, originally isolated from the jellyfish
Aequorea victoria
has become an important tool to study gene expression. However, the sensitivity of wild-type GFP has been below that of standard reporter molecules. Thus, to improve the detection of GFP in transfected cells GFP derivatives have been developed, such as chromophore mutations and codon optimization for mammalian cells that make the protein e.g. several times brighter than wild-type GFP (see Zhang G. et. al.,
An enhanced green fluorescent protein allows sensitive detection of gene transfer in mammalian cells,
Biochem. Biophys. Res. Commun. 227 (3), 707-711 (1996), WO 97/126333 to the University of Florida Research Foundation, Inc. and WO 97/42320 to The Government of the United States of America).
Despite the proven utility of GFP as a reporter molecule for transient gene expression, there still exist problems with the generation of stably transfected cells giving bright signals (see Hanazono Y. et al.,
Green Fluorescent Protein Retroviral Vectors: Low Titer and High Recombination Frequency Suggest a Selection Disadvantage
, Human Gene Therapy 8, 1313-1319 (1997)). The retroviral based method described by Hanazono et al. uses G 418 as a selective drug to isolate stable GFP expressing retroviral producer cell clones, however, the described method was not successful. Gubin A. N. et al. report the successful stable transfection of CHO cells with plasmid DNA encoding both GFP and neomycin phosphor transferase (neo) cassettes (see Gubin A. N. et al.,
Long term, stable expression of green fluorescent protein in mammalian cells
, Biochem. Biophys. Res. Commun., 236(2), 347-350 (1997)). One cloned cell line was reported to maintain “high-level” green fluorescence for 18 weeks in G 418 and another 12 weeks in non-selective medium. No growth disadvantage in mammalian cells by GFP expression was observed. Stable GFP expression was also obtained in murine cells (see Lybarger L. et al.,
Rapid Generation and Flow Cytometric Analysis of Stable GFP
-
Expressing Genes
, Cytometry 25, 211-220 (1996)).
The disadvantage of these stably GFP expressing cell lines of the state of the art is their low GFP expression, making them unsuitable for many applications.
BRIEF SUMMARY OF THE INVENTION
A general goal of the present invention is to provide cells, cell lines and/or cell strains, stably expressing fluorescent proteins, that are e.g. producible by the method also being an object of the present invention.
Further objects of the present invention are methods for process development and process optimization wherein the cells/cell lines/cell strains of the present invention are used.
In the following text the term “cells” will be used for cells as well as cell lines and cell strains.
The cells of the present invention are characterized by a fluorescence signal, in particular a fluorescence signal that is at least 100 times the auto-fluorescence of the respective host cell, although preferably at least 200 times higher than said auto-fluorescence and much preferably at least 1000 times said auto-fluorescence. Preferred cells of the present invention are cells, preferably CHO or HEK cells, genetically modified with humanized GFP, in particular those with a fluorescence about 1000 times higher than the auto-fluorescence of the cell.
Suitable proteins for the present invention, herein referred to as “fluorescent proteins”, are all optically measurable proteins that can be measured without specific activation, whereby GFP and its derivatives are preferred proteins.
The process for obtaining cells according to the present invention is characterized by a transfection step that is performed in a way to ensure transient transfection efficiencies of at least 25% positive cells, although preferably about 30% or more, and much preferably 50% or more positive cells after 1 day. The term transient transfection, as it is used in connection with the present invention means an initial state of transfection that is a first step to stable transfection, whereby only a small percentage of transiently transfected cells reach the stable state.
The cells of the present invention are—due to their specific features—a very useful tool in process development and optimization making such development and optimization much faster. The fluorescent signal of e.g. GFP can quickly and easily be measured with a standard fluorometer. The signal can be correlated with biomass and/or cell number, extending the application of fluorescent-protein-expressing cells to assays where cell growth is of importance. Information thus obtained allows rapid screening and optimization.


REFERENCES:
patent: 5484720 (1996-01-01), Wurm et al.
patent: 5686263 (1997-11-01), Wurm
patent: 5759765 (1998-06-01), Harris et al.
patent: 5874304 (1999-02-01), Zolotukhin et al.
patent: 5968738 (1999-10-01), Anderson et al.
patent: WO 97/26333 (1997-07-01), None
patent: WO 97/42320 (1997-11-01), None
Ropp et al., “Aequorea Green Fluorescent Protein Analysis by Flow Cytometry” Cytometry vol. 21, pp. 309-317, 1995.*
Hawley-Nelson et al., “LIPOFECTAMINE™ Reagent: A New, Higher Efficiency Polycationic Transfection Reagent” Focus vol. 15(3), p. 73-79, 1993.*
Hawley-Nelson et al., “Lipofectamine Reagent: A new Higher Efficiency Polycationic Liposme Transfection Reagent” Focus vol. 15, pp. 309-317, 1993.*
Gubin, A.N. et al., “Long-Term, Stable Expression of Green Fluorescent Protein in Mammalian Cells,”Biochemical and Biophysical Research Communications, 236(2):347-350 (1997).
Hanazono, Y. et al., “Green Fluorescent Protein Retroviral Vectors: Low Titer and High Recombination Frequency Suggest a Selective Disadvantage,”Human Gene Therapy, 8:1313-1319 (1997).
Lybarger, L. et al., “Rapid Generation and Flow Cytometric Analysis of stable GFP-Expressing Cells,”Cytometry, 25:211-220 (1996).
Wurm, F.M. et al., “Gene Transfer and Amplification in CHO Cells,” inRecombinant DNA Biotechnology III: The Integration of Biological and Engineering Sciences, vol. 782 of the Annals of the New York Academy of Sciences p. 70-78 (1996).
Wurm, F.M. et al., “Retrotargeting: Use of Defective Retroviral DNA Fragments to Improve Recombinant Protein Production in Mammalian Cells,” inAnimal Cell Technology: Products of Today, Prospects For Tomorrow, Spier, R.E. et al., (Eds.), ESACT, Butterworth-Heinemann, Oxford, pp. 24-29 (1994).
Zhang, G. et al., “An Enhanced Green Fluorescent Protein Allows Sensitive Detection of Gene Transfer in Mammalian Cells,”Biochemical Biophysical Research Communications, 227(3):707-711 (1996).
Doevendans et al., “The Utility of Fluorescent in Vovo Reporter Genes in Molecular Cardiology,”Biochemical and Biophysical Research Communications, 222(2):352-358 (1996).
Prasher, “Using GFP to see the light,”Trends in Genetics, 11(8):320-323 (1995).

Affiliated with

Also associated with

Rating

Cells, genetically modified with optical marker does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Cells, genetically modified with optical marker, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Cells, genetically modified with optical marker will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3040089