Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
1999-04-23
2001-11-13
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S006120, C435S320100, C435S325000, C536S023200, C536S024100
Reexamination Certificate
active
06316181
ABSTRACT:
DESCRIPTION
Abbreviations
GFP, green fluorescent protein; FACS, fluorescence activated cell sorting; PHAL,
Phseolus vulgaris
leucoagglutinin; DMEM Dulbecco's Modified Eagle Medium; FBS, fetal bovine serum; EDTA, ethylenediamine tetraacetic acid; PBS, phosphate buffered saline; IRES, internal ribosome entry sequence of the encephalomyocarditis virus; CMV, cytomegalovirus; NEO, neomycin.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention generally relates to the establishment of cell lines which are stably transfected with a detectable marker. In particular, the invention provides a DNA vector carrying the GFP marker which can be used to establish stably transfected cell lines; to use for monitoring cell movement, proliferation, and location in vivo; and the invention pertains to stably transfected cell lines which include the GFP marker.
2. Background of the Invention
The ability to transfect cells with exogenous DNA and to monitor such genetically engineered cells in vivo is of fundamental importance to many areas of scientific investigation and emerging medical treatments. For example, ex vivo gene therapy for several diseases depends on the ability to transfect cells. In the treatment of Parkinson's disease, it is possible to surgically remove neural progenitor cells from the patient, grow them in culture, insert therapeutic genes, and then replace the transfected cells back into the patient's brain. However, the ability to monitor correct cell placement and gene expression, which would be a valuable addition to this treatment, is currently not available. Similarly, there is much interest in developing new cancer diagnostic tools to monitor tumor cell invasion,
There are many ways to transfect cells with DNA. These include chemical transfections with lipids or lipid-type compounds, and shocking the cells by either chemical or electrical means. Another means of transfecting DNA into a cell is by using viral vectors (e.g. adenoviral and retroviral vectors) to transfect/transduce the host cells. However, with any of these techniques, the transfection of cells (whether mammalian, plant, or other type of cell) is usually transient, precluding long-term expression of the exogenous DNA or its gene products. For example, the use of viruses and dyes used to monitor cells are limited in that with subsequent cell divisions the ability of daughter cells to express the target DNA or dye diminishes with each cell division.
It would be highly desirable to have available a technique which would provide for the stable tranfection of cells. One important use of such stably transfected cells would be to monitor biological events in “real time”. For example, it is currently not possible to detect the migration of cells such as brain tumor cells or neuronal stem cells. The provision of cells stably transformed with markers which allowed monitoring of biological events in real time would be highly advantageous.
One specific example of an area of medicine that would benefit from the ability to monitor real-time biological events is the field of brain tumor research, such as that involving glioblastoma multiforme, the most common primary brain tumor. Despite significant improvements in the diagnosis and treatment of patients with glioblastoma multiforme, it remains incurable. A key feature that underlies the malignant behavior of this disease is the ability of glioma cells to aggressively infiltrate surrounding brain tissue. Thus, understanding the basic biology of tumor cell invasion/migration may aid in the development of more effective forms of treatment [1-6].
Much information about glioma cell invasion has been gained from studies using a variety of in vivo and in viro models. Tumor cells have been labeled using
Phaseolus vulgars
leucoagglutinin (PHAL), cell labeling dyes, such as fast blue and transfection with the lacZ gene. Each technique has advantages and disadvantages [2, 7, 8 and references therein]. One disadvantage of using externally labeled cells is that long term studies (i.e. lasting multiple cell divisions) are difficult or impossible due to a decrease in signal over time. Although transfection with lacZ is stable, it requires post-processing using a chromogenic substrate for the &bgr;-galactosidase marker enzyme. Caution must be exercised when introducing dyes and substrate reagents into cells because this treatment can affect biological processes such as cell proliferation and motility [9, 10], the very processes one would like to observe.
It would be a distinct advantage to have available an improved means to monitor cell motility in general, and tumor cell migration in particular, e.g. to easily and rapidly identify tumor cells that are in the process of migrating, and to enable examination of their biochemical properties.
SUMMARY OF THE INVENTION
According to the present invention, cell lines have been stably transfected with a green fluorescent protein marker and used to monitor the migration of brain tumor cells. The vector that was used was the pIRES/EGFP vector. This vector was constructed from the commercially available vector pIRES
FIG. 7
) by inserting a gene coding for the green fluorescent protein (GFP) into the multiple cloning site of pIRES. The gene for GFP was obtained from the commercially available vector EGFP FIG.
8
). These vectors and the details of the construction of pIRES/EGFP vector are further described in the Example section under Methods (DNA Constructs).
It is an object of this invention to provide a DNA vector capable of stably transforming cells. In a specific embodiment of the present invention, the vector is the construct pIRES/EGFP, and the form of the GFP protein that is provided is that which is in commercially available EGFP. However, the present invention also comprehends various other vectors which are derivatives of pIRES/EGFP. For example, it is a further object of the present invention to provide vectors with other mutational forms of GFP, such as a form with differing excitation and emission characteristics. Any suitable form of GFP which retains properties allowing it to be monitored after transfection into a cell may be used in the practice of the present invention.
The present invention also comprehends modifications of the vector. For example, another gene of interest may be ligated into the vector so that the expression of the gene of interest could be monitored simultaneously with monitoring cell motility. Or, inducible control elements may be added to the vector which confer the ability to control expression of the GFP protein. Any suitable modification of the vector which retains the ability of the cell transfected with the vector to be monitored in real time by tracking GFP fluorescence may be used in the practice of the present invention.
It is a further object of the present invention to provide cell lines which have been stably transformed with the vector pIRES/EGFP. In particular, the cell lines 3RT1 and U373GFP are provided by the instant invention. While these cell lines are of rat and human derivation, cell lines derived from any source (for example, plants) may be used in the practice of the present invention.
It is an object of the present invention to provide cell lines stably transfected with a green fluorescent protein as reagents for asessing tumor cell migration. It is a further object of the present invention to provide the cell lines transfected with a green fluorescent protein to be utilized for the real-time assessment of other biological processes and events. Those of skill in the art will recognize that the cell lines of the present invention may be utilized for the assessment of any suitable biological process or event.
It is an object of the present invention to provide glioma cells which have been stably transfected with the vector pIRES/EGFP. It is a further object of the present invention to provide a method for stably transfecting other cell types with the vector pIRES/EGFP. For example, neuronal stem cells from both human and rat co
Broaddus William C.
Fillmore Helen
Gillies George T.
Shurm, Jr. John S.
Brusca John S.
McGuireWoods LLP
Virginia Commonwealth University
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