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

a ribonucleic acid sequence and cell line technology, applied in the field of nucleic acid probes, can solve the problems of promising application of this line of research to in-vivo detection, and achieve the effect of inhibiting the proteolytic activity of the protease and being readily measured

Inactive Publication Date: 2006-07-06
CHROMOCELL CORP
View PDF20 Cites 14 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[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

Problems solved by technology

The results though discernible are barely so and the applicabilit

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Examples

Experimental program
Comparison scheme
Effect test

example

[0173]1

[0174] General Protocol. Starting Material: Molecular Beacons may be introduced into cells which are not expressing any RNAs from plasmids, or they may be used to detect RNA messages encoded from plasmids. The method of introduction of the beacons into either of these two types of cells is identical. The protocol below only requires that the cells to be analyzed are separable from each other and are amenable to FACS analysis.

[0175] 1) As described more thoroughly in the description of the invention, beacons can be used in conjunction with FACS to sort out cells of a tissue based on expression or lack of expression within cells of specific RNAs. To this end, cells must first be separated from each other by standard and well established methods such as homogenization and further chemical treatment. Appropriate beacons may then be introduced into such cells according to the protocol below.

[0176] 2) Second, one may use beacons to select for cells expressing particular RNAs enco...

example 2

Selection of Cells Using Beacons

[0178] 1) Transfect beacons into cells: Beacons must be designed such that they will recognize the desired RNA either by hybridizing to a sequence endogenous in the RNA or by hybridizing to a tag that is added to the native RNA sequence. The design of beacons is elaborated upon in the description of the invention.

[0179] Transfection may be carried out by a vast variety of methods, similar to the transfection of plasmids into cells. The method employed should be chosen based on the cell type being used as some cells respond better to some transfection methods over other methods. Transfection should be performed according to the instructions of the manufacturer of the transfection reagent used.

[0180] Transfection of beacons into cells may be carried out either on cells in suspension or on cells growing on solid surfaces, depending on the transfection reagent used.

[0181] 2) Following the transfection of beacons into cells, the cells may then be subje...

example 3

Generation of Cell Lines Expressing One or More RNAs

[0182] Following FACS selection, the positive-scoring cells can be maintained in appropriate medium as described in more detail in the description of the invention. This cells would give rise to cell lines expressing the RNAs of interest.

[0183] Concentration of the Beacon: The concentration of beacon to be used depends on several factors. For instance, one must consider the abundance within cells of the RNA to be detected and the accessibility of this RNA to the beacon. For instance, if the RNA to be detected is present in very low amounts or if it is found in a portion of the RNA which is not readily accessible based on the three-dimensional folding of the RNA, then more beacon should be used here then in cases where the RNA to be detected is in high abundance and where the site recognized by the beacon is fully accessible. The exact amount of beacon to be used will have to be determined empirically for each application.

[0184] ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
Timeaaaaaaaaaa
Electrical resistanceaaaaaaaaaa
Antisenseaaaaaaaaaa
Login to view more

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 ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12Q1/68C07H21/02A01K67/027C07H21/04C12N5/00C12N5/10C12N15/00C12N15/09C12N15/15C12N15/63
CPCC07H21/04C12N15/63C12Q1/68C12Q1/6809C12Q1/6818C12Q1/6841C12Q2600/158G01N2500/04C12Q2565/601C12Q2525/301C12Q2537/143C12Q2565/1015C12Q2527/127C12Q2521/537C12N15/09C12N15/00C12N15/11C12N15/85
Inventor SHEKDAR, KAMBIZBLOBEL, GUNTER
Owner CHROMOCELL CORP
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap