Isolation of living cells and preparation of cell lines based on detection and quantification of preselected cellular ribonucleic acid sequences

Abstract

The invention is directed to reliable and efficient detection of mRNAs as well as other RNAs in living cells and its use to identify and, if desired, separate cells based on their desired characteristics. Such methods greatly simplify and reduce the time necessary to carry out previously-known procedures, and offers new approaches as well, such as selecting cells that generate a particular protein or antisense oligonucleotide, generating cell lines that express multiple proteins, generating cell lines with knock-out of one or more protein, and others.

Description

CROSS-REFERENCE TO RELATED APPLICATION [0001] This application claims priority to provisional application Ser. No. 60 / 166,987, filed Nov. 23, 1999, incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION [0002] 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. [0003] This property has enabled researchers to detect specific nucleic acids ...

AI Technical Summary

Benefits of technology

[0010] 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.

[0049] In the foregoing aspect of the invention, the at least two DNA sequences may reside on the same construct. The first and the second epitope tags may each independently be in frame or out of frame with the protein. The FACS may be used to isolate cells expressing both constructs by isolating the cells expressing the strongest fluorescence. As with the previous methods described herein, the transfection may be done simultaneously or sequentially. Selection when the same drug resistance marker is present in both constructs and simultaneous transfection is performed may be achieved by increasing the level of the drug.

[0064] In another aspect, the present invention is directed to a proteolytic activity-generating unitary hybridization probe, herein referred to as a probe protease. Such probe proteases operate in a similar fashion to a molecular beacon, but instead of a change in fluorescence on interaction of the beacon with its target nucleic acid sequence, the probe protease becomes proteolytic in the presence of the target. The probe protease composition provides at one end of a molecular beacon-type oligonucleotide a protease, and at the other, a complementary protease inhibitor. When the oligonucleotide is not hybridized to a target sequence, the proximity of the ends of the oligonucleotide permit the protease and the protease inhibitor to interact, inhibiting proteolytic activity of the protease. However, upon hybridization of the target sequence of the oligonucleotide to the target, the protease and its inhibitor are separated, activating the protease. The activity of the protease can be readily measured, and furthermore, the active protease in the presence of a particular nucleic acid target sequence may be employed not only for detection purposes but also for therapeutic purposes, in which, for example, a cell in which the probe protease is delivered is proteolyzed and rendered nonviable if a particular gene is transcribed, for example, one related to cellular transformation, oncogenesis, dysproliferation, and the like.

Problems solved by technology

The results though discernible are barely so and the applicability of this line of research to in-vivo detection was not promising.