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.

1 . A method for generating a cell line expressing at least one preselected protein comprising the steps of:
 a) transfecting cell lines with at least one DNA construct encoding said 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 DNA construct and the at least one drug resistance marker;   c) exposing said selected cells to a first molecular beacon that fluoresces upon hybridization to an RNA transcript of said at least one preselected protein;   d) isolating said cells that fluoresces;   e) generating a cell line expressing said at least one preselected protein by growing said isolated cells.   
   
   
       2 . The method of  claim 1  wherein isolating said cells that fluoresce is carried out using fluorescence activated cell sorter technology. 
   
   
       3 . The method of  claim 1 , wherein said cell line expresses a second preselected protein, said steps further including transfecting said cell line with a second DNA construct encoding a second preselected protein and a drug resistance marker; selecting for cells expressing said second marker; exposing said cells to a second molecular beacon which fluoresces upon hybridization to an RNA transcript of said second preselected protein and isolating cells that exhibit fluorescence of each of said at least one and said second mRNA transcripts. 
   
   
       4 . The method of  claim 3  wherein said step is performed either simultaneously or sequentially. 
   
   
       5 - 11 . (canceled) 
   
   
       12 . A method 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 said 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 DNA construct and the at least one drug resistance marker;   c) exposing said cells to a molecular beacon that fluoresces upon hybridization to said antisense RNA molecule;   d) isolating said cells that fluoresce; and   e) generating a cell line expressing said preselected antisense RNA sequence by growing said isolated cells.   
   
   
       13 . The method of  claim 12  wherein isolating said cells that fluoresce is carried out using fluorescence activated cell sorter technology. 
   
   
       14 . The method of  claim 12 , wherein said cell line expresses at least one second preselected antisense RNA molecule, said steps further including transfecting said cell line with a second DNA construct encoding a second preselected antisense RNA molecule and a second drug resistence marker; selecting for cellular expressing said second marker; exposing said cells to a second molecular beacon which fluoresces upon hybridization to a said second antisense RNA molecule; and isolating cells that exhibit fluorescence of each of said at least one and said second mRNA transcripts. 
   
   
       15 - 21 . (canceled) 
   
   
       22 . A method for quantifying the level of at least one RNA transcript expression in a biological sample comprising the steps of
 a) exposing said biological sample to a first molecular beacon which fluoresces upon hybridization with said RNA transcript;   b) quantitating the level of fluorescence in said biological sample; and   c) correlating said level of fluorescence with said level of said at least one mRNA transcript.   
   
   
       23 . The method of  claim 22 , wherein said biological sample is selected from the group consisting of cellular sample and a tissue sample. 
   
   
       24 . The method of  claim 22  wherein said RNA transcript is selected from the group consisting of RNA that encodes a protein, a structural RNA, and an antisense RNA. 
   
   
       25 . The method of  claim 22 , wherein said biological sample is fixed. 
   
   
       26 . The method of  claim 22 , wherein said fluorescence is quantitated by fluorescence microscopy or fluorescence-activated cell sorter technology. 
   
   
       27 . (canceled) 
   
   
       28 . The method of  claim 22  wherein said cells expressing said RNA are isolated using fluorescence cell sorter technology. 
   
   
       29 - 34 . (canceled) 
   
   
       35 . A method for generating cells functionally null for expression of at least one preselected protein comprising the steps of providing in said cells a plurality of antisense RNAs to said preselected protein, each provided in accordance with  claim 12 , wherein said plurality of antisense RNA to said at least one preselected protein binds essentially all mRNA transcripts of said at least one preselected protein. 
   
   
       36 . The method of  claim 35 , wherein said preselected protein is an alternatively spliced form of a gene product. 
   
   
       37 . A method for generating a transgenic animal that is a functionally null-expressing mutant of at least one preselected protein comprising carrying out the steps of  claim 35  utilizing embryonic stem cells, determining the viability of said stem cells functionally null expressing said preselected protein, and using said viable embryonic stem cells to produce said transgenic animal. 
   
   
       38 . (canceled) 
   
   
       39 . A method for generating a cell line expressing a lethal antisense RNA under control of an inducible promoter by carrying out the method of  claim 12  wherein step (a) is performed in the presence of a minimal amount of an inducer. 
   
   
       40 - 43 . (canceled)