Claims for Patent: 7,449,179

Summary for Patent: 7,449,179

Title:	Vectors for conditional gene inactivation
Abstract:	A method of preparing gene trapping libraries, and gene targeted cells for conditional inactivation of genes, is provided. The invention provides a vector having a mutational element cassette and a gene trap cassette, each cassette having site-specific recombination sequences.
Inventor(s):	Xin; Hong-Bo (Ithaca, NY), Kotlikoff; Michael (Ithaca, NY)
Assignee:	Cornell Research Foundation, Inc. (Ithaca, NY)
Application Number:	10/416,995
Patent Claims:	1. A genetically engineered vector comprising: a) a mutational element cassette comprising operably linked: i) a DNA comprising a first site-specific recombination sequence for a first recombinase; ii) a DNA comprising a mutational sequence which comprises a splice acceptor sequence linked to a first marker gene linked to a polyadenylation sequence; and iii) a DNA comprising a second site-specific recombination sequence for the first recombinase, wherein the first and second site-specific recombination sequences in a)i and a)iii are mutant sequences that are substrates for the first recombinase; and b) a gene trap cassette comprising operably linked: i) a DNA comprising a first site-specific recombination sequence for a second recombinase; ii) a DNA comprising a first gene trap element comprising a promoter operably linked to a second marker gene operably linked to a splice donor sequence and a second gene trap cassette comprising promoter operably linked to a unique sequence not present in the genome of a selected host cell; and iii) a DNA comprising a second site-specific recombination sequence for the second recombinase; wherein the gene trap cassette is 3' to the mutational element cassette, wherein the first and second recombinases are different, wherein the DNA of a)i) and the DNA of a)iii) are in opposite orientation to each other, and wherein the DNA of b)i) and the DNA of b)iii) are in the same orientation, wherein if the vector inserts into an exon in the genome of the cell and is expressed, the unique sequence is present in a RNA transcript but if the vector inserts into an intron in the genome of the cell and is expressed, the unique sequence is not spliced into mRNA. 2. The vector of claim 1 wherein DNA comprising the mutational sequence is inverted relative to the DNA of b)ii). 3. The vector of claim 1 wherein the second marker gene is a selectable marker gene. 4. The vector of claim 1 wherein the DNA of a)i) and the DNA of a)iii) are lox sequences. 5. The vector of claim 1 wherein the DNA of b)i) and the DNA of b)iii) are flip recombinase recognition sequences (FRT) sequences. 6. The vector of claim 5 further comprising a 5' LTR and a 3' LTR and a third site-specific recombination sequence for the second recombinase, wherein the 3' LTR is 5' to the mutational element cassette and the 5' LTR is 3' to the gene trap cassette, wherein the third site-specific recombination sequence is 3' to the 3' LTR and 5' to the DNA of a)i) and is in an orientation that is inverted relative to the DNA of b)i) and b)iii), wherein the DNA of a)ii) is in the same orientation as the DNA of b)ii), and wherein the LTRs are positioned so that transcription from the 5' LTR is in the opposite orientation as transcription from the promoters in the DNA of b)ii). 7. The vector of claim 1 wherein the mutational sequence further comprises an internal ribosome entry site operatively positioned between the splice acceptor sequence and the initiation codon of the second marker gene. 8. The vector of claim 1 wherein the first or the second marker gene is selected from the group consisting of a marker conferring antibiotic resistance, an enzymatic marker, and a fluorescently detectable marker. 9. The vector of claim 8 wherein the first marker gene is green fluorescent protein (Gfp) or LacZ. 10. The vector of claim 8 wherein the second marker gene encodes neomycin or puromycin resistance. 11. The vector of claim 1 further comprising a DNA sequence which is 5' to the mutational element cassette and comprises a multiple cloning site. 12. The vector of claim 1 wherein the DNA of a)i) and the DNA of a)iii) are mutant lox sequences and the DNA of b)i) and the DNA of b)iii) are FRT sequences. 13. A method of gene trapping comprising: a) introducing a vector into a mammalian cell in vitro to yield genetically altered mammalian cells, wherein the vector comprises: i) a mutational element cassette comprising operably linked: a DNA comprising a first site-specific recombination sequence for a first recombinase; a DNA comprising a mutational sequence which comprises a splice acceptor sequence linked to a first marker gene linked to a polyadenylation sequence; and a DNA comprising a second site-specific recombination sequence for the first recombinase, wherein the first and second site-specific recombination sequences in a)i and a)iii are mutant sequences that are substrates for the first recombinase; and ii) a gene trap cassette comprising operably linked: a DNA comprising a first site-specific recombination sequence for a second recombinase; a DNA comprising a first gene trap element comprising a promoter operably linked to a second marker gene operably linked to a splice donor sequence and a second gene trap cassette comprising promoter operably linked to a unique sequence; and a DNA comprising a second site-specific recombination sequence for the second recombinase; wherein the gene trap casseffe is 3' to the mutational element cassette, wherein the first and second recombinases are different, wherein the DNA comprising the first site-specific recombination sequence for the first recombinase and the DNA comprising the second site-specific recombination sequence for the first recombinase are in opposite orientation to each other, and wherein the DNA comprising the first site-specific recombination sequence for the second recombinase, and the DNA comprising the second site-specific recombination sequence for the second recombinase are in the same orientation, wherein if the vector inserts into an exon in the genome of the cell and is expressed, the unique sequence is present in a RNA transcript but if the vector inserts into an intron in the genome of the cell and is expressed, the unique sequence is not spliced into mRNA; and b) identifying at least one first genetically altered cell, the genome of which comprises the vector and expresses the second marker gene. 14. The method of claim 13 wherein the introduction of the vector to the cell is via electroporation, viral infection, retrotransposition, microinjection, or transfection. 15. The method of claim 13 further comprising isolating the first genetically altered cell. 16. The method of claim 13 wherein the mammalian cell is a murine, rat or human cell. 17. The method of claim 13 wherein the first genetically altered cell is a rodent embryonic stem cell. 18. The method of claim 13 further comprising c) introducing to the first genetically altered cell the second recombinase to yield a second genetically altered cell, the genome of which has undergone a recombination event relative to the first genetically altered cell such that the genome of the second genetically altered cell does not comprise the gene trap element and one of the site-specific recombination sequences for the second recombinase. 19. An isolated second genetically altered cell prepared by the method of claim 18. 20. The isolated second genetically altered cell of claim 19 which is a rodent embryonic stem cell. 21. The method of claim 13 wherein the DNA of a)i) and the DNA of a)iii) are mutant lox sequences and the DNA of b)i) and the DNA of b)iii) are FRT sequences. 22. The method of claim 13 wherein the mutational cassette of a) is inverted relative to the gene trap cassette of b).

Details for Patent 7,449,179

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