Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-11-22
2004-02-17
Ketter, James (Department: 1635)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C536S023100, C536S023200, C536S023400, C536S023500, C536S024100, C536S024200, C536S024300, C536S024310, C536S024320, C424S093210, C424S093700, C435S320100, C435S471000
Reexamination Certificate
active
06692965
ABSTRACT:
BACKGROUND OF THE INVENTION
A molecular beacon is a nucleic acid probe that recognizes and reports the presence of a specific nucleic acid sequence. The probes are hairpin-shaped sequences with a central stretch of nucleotides complementary to the target sequence, and termini comprising short mutually complementary sequences. One terminus is covalently bound to a fluorophore and the other to a quenching moiety. When in their native state with hybridized termini, the proximity of the fluorophore and the quencher is such that no fluorescence is produced. The beacon undergoes a spontaneous fluorogenic conformational change when hybridized to its target nucleic acid. See, for example, U.S. Pat. No. 5,925,517.
This property has enabled researchers to detect specific nucleic acids primarily in in vitro reactions and in some cases even in living cells. In-situ visualization of messenger RNA has been achieved using molecular beacons delivered to living-cells in liposomes (Matsuo, 1998, Biochim. Biophys. Acta 1379:178-184). Studies involving cells have to date employed beacons generated with the very weak fluorophore EDANS as this was the only one known to be quenched by the quencher EDAC. The results though discernible are barely so and the applicability of this line of research to in-vivo detection was not promising.
SUMMARY OF THE INVENTION
In one aspect, the present invention is directed to a method for generating a cell line expressing at least one preselected protein comprising the steps of:
a) transfecting a cell line with at least one DNA construct encoding at least one preselected protein and at least one drug resistance marker;
b) selecting for cells resistant to a drug to which said marker confers resistance, said cells transcribing the at least one drug resistance marker;
c) exposing the selected cells to a first molecular beacon that fluoresces upon hybridization to an RNA transcript of the at least one preselected protein;
d) isolating the cells that fluoresces;
e) generating a cell line expressing the at least one preselected protein by growing the isolated cells.
The aforementioned method may be carried out using fluorescence activated cell sorter technology. In a further aspect, the cell line may be made to express a second preselected protein by carrying out further steps including transfecting the cell line with a second DNA construct encoding a second preselected protein and a second drug resistance marker; selecting for cells expressing the second marker; exposing the cells to a second molecular beacon which fluoresces upon hybridization to an RNA transcript of the second preselected protein and isolating cells that exhibit fluorescence of each of the at least one and the second mRNA transcripts. Cell lines expressing more than two proteins may be provided by repeating the steps. The method to introduce the second protein may be performed either simultaneously or sequentially. If the first and second drug resistance markers are the same, simultaneous selection may be achieved by increasing the level of the drug.
In another aspect of the invention, a method is provided for generating a cell line expressing at least one preselected protein comprising the steps of:
a) transfecting a cell line with at least one DNA construct encoding the at least one preselected protein, at least one drug resistance marker and at least one first epitope tag;
b) selecting for cells resistant to a drug to which said marker confers resistance, said cells transcribing the at least one drug resistance marker;
c) exposing the cells to a first molecular beacon that fluoresces upon hybridization with the RNA transcript of the first epitope tag;
d) isolating the cells that fluoresce; and
e) generating a cell line expressing the at least one preselected protein by growing the isolated cells.
The aforementioned method may be carried out using a fluorescence activated cell sorter technology. The DNA portion of the construct encoding the epitope tag may be in frame or out of frame with the portion of the DNA construct encoding the at least one protein. In a further embodiment, the cell line may be made to express at least a second preselected protein; the steps further including transfecting the cell line with a second DNA construct encoding the second preselected protein, a second drug resistance marker and a second epitope tag, selecting for cells transcribing the second marker; exposing the cells to a second molecular beacon that fluoresces upon hybridization with the RNA transcript of the second epitope tag, and isolating the cells that exhibit fluorescence of each of the first and the second epitope tag. In the case of two proteins, the portion of the DNA sequence encoding the second epitope tag may also be in frame or out of frame with the portion of the DNA sequence encoding the second protein. The second preselected protein may be transfected either simultaneously or sequentially with the first. Should the method be performed simultaneously, and the same drug resistance marker used for both constructs, a higher level of drug may be used to select for cells expressing both constructs. Furthermore, more than two proteins may be provided in the cell line by repeating the aforementioned steps.
In yet a further aspect of the present invention, a method is provided for generating a cell line expressing at least one preselected antisense RNA molecule comprising the steps of:
a) transfecting a cell line with a DNA construct encoding the at least one preselected antisense RNA molecule and at least one drug resistance gene;
b) selecting for cells resistant to a drug to which said marker confers resistance, said cells transcribing the at least one drug resistance marker;
c) exposing the cells to a molecular beacon that fluoresces upon hybridization to the antisense RNA molecule;
d) isolating the cells that fluoresce; and
e) generating a cell line expressing the preselected antisense RNA sequence by growing the isolated cells.
Isolating the cells that fluoresce may be carried out using fluorescence activated cell sorter technology. The cell line may be made to express at least one second preselected antisense RNA molecule, the steps further including simultaneously or sequentially transfecting the cell line with a second DNA construct encoding a second preselected antisense RNA molecule and a second drug resistence marker; selecting for cellular expressing the second marker; exposing the cells to a second molecular beacon which fluoresces upon hybridization to a the second antisense RNA molecule; and isolating cells that exhibit fluorescence of each of the at least one and the second mRNA transcripts. If the method is performed simultaneously and the same drug resistance marker is used in both constructs, selection can be achieved using a higher level of drug. More than two antisense RNA molecules may be provided by repeating the aforementioned steps with another construct.
In yet another aspect of the invention, a method is provided for generating a cell line expressing at least one preselected antisense RNA molecule comprising the steps of:
a) transfecting a cell line with a DNA construct encoding the at least one preselected antisense RNA molecule, at least one drug resistance marker and a first epitope tag nucleotide sequence;
b) selecting for cells resistant to a drug to which said marker confers resistance, said cells transcribing the at least one drug resistance marker;
c) exposing the selected cells to a first molecular beacon that fluoresces upon hybridization to a mRNA transcript of the first epitope tag;
d) isolating the cells that fluoresce; and
e) generating a cell line expressing the at least one preselected antisense RNA molecule by growing the isolated cells.
The cell line may be made to express at least one second preselected epitope tagged antisense RNA, the steps further including transfecting the cell line with a second DNA construct encoding a second preselected epitope-tagged antisense RNA molecule a second drug resistance marker; selecting for cellular expressing the second mark
Blobel Gunter
Shekdar Kambiz
Chromocell Corporation
Fish & Neave
Gunnison Jane T.
Ketter James
Su Li
LandOfFree
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