Claims for Patent: 5,652,128

Summary for Patent: 5,652,128

Title:	Method for producing tagged genes, transcripts, and proteins
Abstract:	The invention described here is a method whereby a molecular tag is put on a gene, transcript and protein in a single recombinational event. The protein tag takes the form of a unique peptide that can be recognized by an antibody or other specific reagent, the transcript tag takes the form of the sequence of nucleotides encoding the peptide that can be recognized by a specific polynucleotide probe, and the gene tag takes the form of a larger sequence of nucleotides that includes the peptide-encoding sequence and other associated nucleotide sequences. The central feature of the invention in its essential form is that the tag-creating DNA has a structure such that when it is inserted into an intron within a gene it creates two hybrid introns separated by a new exon encoding the protein tag. A major virtue of the method is that it allows one to identify new proteins or protein-containing structures, and, having done so, to readily identify and analyze the genes encoding those proteins.
Inventor(s):	Jarvik; Jonathan Wallace (Pittsburgh, PA)
Assignee:
Application Number:	08/000,619
Patent Claims:	1. A method for tagging genes, transcripts and proteins in eucaryotic cells comprising: (1) producing a tagged gene by inserting a DNA sequence via a known vector into an intron of a gene by: (a) selecting a DNA sequence, said sequence comprising: (i) a 5' portion free of any nucleotide sequence selected from the group consisting of CAGGTAAGT, CAGGTGAGT, AAGGTAAGT, and AAGGTGAGT, followed by; (ii) a nucleotide sequence selected from the group consisting of TACTAAC, TGCTAAC, TCCTAAC, TTCTAAC, TACTGAC, TGCTGAC, TCCTGAC, and TTCTGAC, followed by; (iii) a nucleotide sequence 14 to 34 nucleotides in length followed by; (iv) a nucleotide sequence selected from the group consisting of CAGG and TAGG, followed by; (v) an open reading frame encoding a known peptide tag recognizable by a known reaction characteristic of said known peptide tag, followed by; (vi) a nucleotide sequence selected from the group consisting of CAGGTAAGT, CAGGTGAGT, AAGGTAAGT, and AAGGTGAGT, followed by; (vii) a 3' portion; (b) inserting said DNA sequence into said intron within said gene to create a tagged gene; and (2) incubating said tagged gene within a eucaryotic cell so as to maintain intact or to introduce said tagged gene within the genome of said eucaryotic cell. 2. The method of claim 1 wherein said DNA sequence is introduced into said intron by in vitro recombination methods. 3. The method of claim 1 wherein said DNA sequence is introduced into said intron by in vivo recombination. 4. The method of claim 1 wherein said cell is that of a eucaryotic microorganism. 5. The method of claim 1 wherein said cell belongs to a culture of pleuripotent stem cells derived from a multicellular organism. 6. The method of claim 1 wherein said cell belongs to a somatic cell culture derived from a multicellular organism. 7. The method of claim 1 wherein expression of said gene is promoted by introducing said DNA molecule into said cell by a method chosen from the following group: transformation, electroporation, transfection, bulk loading, liposome fusion. 8. The method of claim 1 wherein said DNA sequence is introduced into said intron by the method of transposon insertion. 9. The method of claim 1 wherein said DNA sequence is part of a recombinant plasmid. 10. The method of claim 1 wherein said DNA sequence is part of a recombinant virus. 11. The method of claim 1 wherein said DNA sequence is part of a recombinant transposon. 12. The method of claim 1 wherein said DNA sequence becomes stably incorporated into the genome of said cell. 13. The method of claim 1 wherein said peptide tag is recognized by specific monoclonal antibodies. 14. The method of claim 1 wherein said peptide tag is recognized by specific polyclonal antibodies. 15. The method of claim 1 wherein said peptide tag is recognized by specific reagents that are not antibodies. 16. A method as set forth in claim 1 wherein said gene is contained in a living cell. 17. A method as set forth in claim 1 wherein said gene is contained in a library of cloned DNA. 18. A method as set forth in claim 1 wherein said gene is contained in a DNA clone. 19. A method as set forth in claim 1 wherein said gene is contained in isolated genomic, viral or organelle DNA. 20. The method of claim 1 wherein said DNA sequence is comprised of two segments, each of said segments comprising: (i) a 5' portion free of any nucleotide sequence selected from the group consisting of CAGGTAAGT, CAGGTGAGT, AAGGTAAGT, and AAGGTGAGT, followed by; (ii) a nucleotide sequence selected from the group consisting of TACTAAC, TGCTAAC, TCCTAAC, TTCTAAC, TACTGAC, TGCTGAC, TCCTGAC, and TTCTGAC, followed by: (iii) a nucleotide sequence of 14 to 34 nucleotides in length, followed by; (iv) a nucleotide sequence selected from the group consisting of CAGG and TAGG, followed by; (v) an open reading frame encoding a known peptide tag recognizable by a known reaction characteristic of said known peptide tag, and followed by; (vi) a nucleotide sequence selected from the group consisting of CAGGTAAGT, CAGGTGAGT, AAGGTAAGT, and AAGGTGAGT; and wherein said two segments are oriented in opposite directions. 21. The method of claim 20 wherein said two segments share the same reading frame which can be translated in either direction to give distinct peptides. 22. The method of claim 1 wherein said DNA sequence is comprised of multiple segments, each of said segments comprising: (i) a 5' portion free of any nucleotide sequence selected from the group consisting of CAGGTAAGT, CAGGTGAGT, AAGGTAAGT, and AAGGTGAGT, followed by; (ii) a nucleotide sequence selected from the group consisting of TACTAAC, TGCTAAC, TCCTAAC, TTCTAAC, TACTGAC, TGCTGAC, TCCTGAC, and TTCTGAC, followed by; (iii) a nucleotide sequence of 14 to 34 nucleotides in length, followed by; (iv) a nucleotide sequence selected from the group consisting of CAGG and TAGG, followed by; (v) an open reading frame encoding a known peptide tag recognizable by a known reaction characteristic of said known peptide tag, and followed by; (vi) a nucleotide sequence selected from the group consisting of CAGGTAAGT, CAGGTGAGT, AAGGTAAGT, and AAGGTGAGT; and wherein said two segments are oriented in the same direction. 23. A eukaryotic cell containing at least one gene tagged according to the method of claim 1. 24. A method for tagging genes, transcripts and proteins in eucaryotic cells comprising: (1) producing a tagged gene by in vitro ligation of a DNA sequence into a known restriction site within an intron within a cloned gene in a known plasmid or vital vector, by: (a) selecting a DNA sequence, said sequence comprising: (i) a 5' end suitable for ligation into a restriction site; (ii) a nucleotide sequence selected from the group consisting of TACTAAC, TGCTAAC, TCCTAAC, TTCTAAC, TACTGAC, TGCTGAC, TCCTGAC, and TTCTGAC, followed by; (iii) a nucleotide sequence 14 to 34 nucleotides in length, followed by; (iv) a nucleotide sequence selected from the group consisting of CAGG and TAGG, followed by; (v) an open reading frame encoding a known peptide tag recognizable by a known reaction characteristic of said known peptide tag, followed by; (vi) a nucleotide sequence selected from the group consisting of CAGGTAGT, CAGGTAAGT, AAGGTAAGT, and AAGGTGAGT, followed by; (vii) a 3' end suitable for ligation into a restriction site; (b) inserting said DNA sequence into a known restriction site in said intron within a cloned gene in a known plasmid or viral vector to create a tagged gene; and (2) introducing said tagged gene into a eucaryotic cell capable of expressing said gene by a method selected from the group consisting of transformation, electropotation, transfection, bulk loading, and liposome fusion. 25. A method for tagging genes, transcripts and proteins comprising: (1) producing a tagged gene by inserting a DNA sequence resident in a transposon into a cloned gene in vivo, said gene being resident in a vital or plasmid vector, comprising: (a) selecting a DNA sequence, said sequence comprising: (i) a 5' portion free of any nucleotide sequence selected from the group consisting of CAGGTAAGT, CAGGTGAGT, AAGGTAAGT, and AAGGTGAGT, followed by; (ii) a nucleotide sequence selected from the group consisting of TACTAAC, TGCTAAC, TCCTAAC, TTCTAAC, TACTGAC, TGCTGAC, TCCTGAC, and TTCTGAC, followed by; (iii) a nucleotide sequence 14 to 34 nucleotides in length, followed by; (iv) a nucleotide sequence selected from the group consisting of CAGG and TAGG, followed by; (v) an open reading frame encoding a known peptide tag recognizable by a known reaction characteristic of said known peptide tag, followed by; (vi) a nucleotide sequence selected from the group consisting of CAGGTAGT, CAGGTAAGT, AAGGTAAGT, and AAGGTGAGT; (b) inserting said DNA sequence into an existing intron within said gone to create a tagged gene; (2) introducing said tagged gone into a eucaryotic cell capable of expressing said tagged gone by a method chosen from the group consisting of transformation, electropotation, transfection, bulk loading, and liposome fusion; and (3) detecting the expression of said DNA sequence as a peptide recognizable by said known reaction. 26. A method for tagging genes, transcripts and proteins in eucaryotic cells comprising: (1) producing a tagged gene by inserting in vivo a DNA sequence via a known vector into an intron of a gene by: (a) selecting a DNA sequence, said sequence comprising: (i) a 5' portion free of any nucleotide sequence selected from the group consisting of CAGGTAAGT, CAGGTGAGT, AAGGTAAGT, and AAGGTGAGT, followed by; (ii) a nucleotide sequence selected from the group consisting of TACTAAC, TGCTAAC, TCCTAAC, TTCTAAC, TACTGAC, TGCTGAC, TCCTGAC, and TTCTGAC, followed by; (iii) a nucleotide sequence 14 to 34 nucleotides in length, followed by; (iv) a nucleotide sequence selected from the group consisting of CAGG and TAGG, followed by; (v) an open reading frame encoding a known peptide tag recognizable by a known antibody, followed by; (vi) a nucleotide sequence selected from the group consisting of CAGGTAAGT, CAGGTGAGT, AAGGTAAGT, and AAGGTGAGT; (b) introducing said DNA sequence into eucaryotic cells and thence into the genome of said cells in vivo using a method selected from the group consisting of transposon mobilization, transformation, electroporation, transfection, bulk loading, and liposome fusion; followed by (2) immunochemically identifying cells in which said peptide tag is expressed.

Details for Patent 5,652,128

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2039-02-26
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2039-02-26
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2039-02-26