Production of antibodies using Cre-mediated site-specific recomb

Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal

Patent

Rate now

  [ 0.00 ] – not rated yet Voters 0   Comments 0

Details

800 21, 800 25, 800 4, 435354, C12N 500, A61K 4800

Patent

active

060910011

ABSTRACT:
A method to produce a cell expressing an antibody from a genomic sequence of the cell comprising a modified immunoglobulin locus using Cre-mediated site-specific recombination is disclosed. The method involves first transfecting an antibody-producing cell with a homology-targeting vector comprising a lox site and a targeting sequence homologous to a first DNA sequence adjacent to the region of the immunoglobulin loci of the genomic sequence which is to be converted to a modified region, so the first lox site is inserted into the genomic sequence via site-specific homologous recombination. Then the cell is transfected with a lox-targeting vector comprising a second lox site suitable for Cre-mediated recombination with the integrated lox site and a modifying sequence to convert the region of the immunoglobulin loci to the modified region. This conversion is performed by interacting the lox sites with Cre in vivo, so that the modifying sequence inserts into the genomic sequence via Cre-mediated site-specific recombination of the lox sites. Also disclosed are a form of the method used to produce a cell expressing a modified antibody molecule using Cre-mediated site-specific recombination, and antibody-producing cells obtainable by the disclosed methods. Class-switching modifications of human antibodies produced in murine hybridoma cells are exemplified.

REFERENCES:
patent: 4816567 (1989-03-01), Cabilly et al.
patent: 4959317 (1990-09-01), Sauer
patent: 5202238 (1993-04-01), Fell et al.
patent: 5434066 (1995-07-01), Beebe
patent: 5434340 (1995-07-01), Krimpenfort
patent: 5545806 (1996-08-01), Lonberg
Southern & Berg, "Transformation of Mammalian Cells to Antibioic Resistance with a Bacterial Gene Under Control of the SV40 Early Region Promoter," J. Mol. Appl. Genet. 1:327-341 (1982).
Ito et al., "Solid phase synthesis of polynucleotides. VI. Further studies on polystyrene copolymers for the solid support," Nuc. Acids Res. 10:1755-1769 (1982).
Spira et al., "The Identification of Monoclonal Class Switch Variants by Sib Selection and an ELISA Assay," J. Immunological Methods 74:307-315 (1984).
Neuberger et al., "Recombinant antibodies possessing novel effector functions," Nature 312:604-608 (1984).
Blochlinger & Diggelmann, "Hygromycin B Phosphotransferase as a Selectable Marker for DNA Transfer Experiments with Higher Eucaryotic Cells," Mol. Cell Biol., 4:2929-2931 (1984).
Tan et al., "A Human'Mouse Chimeric Immunoglobulin Gene with a Human Variable Region is Expressed in Mouse Myeloma Cells," J. Immunol. 135:3564-3567 (1985).
Waterhouse et al., "Combinatorial infection and in vivo recombination: a strategy for making large phage antibody repertoires," Nucleic Acids Res. 21:2265-2266 (1993).
Steplewski et al., "Isolation and characterization of anti-monodialoganglioside monoclonal antibody 19-9 class-switch variants," Proc. Natl. Acad. Sci. USA 82:8653-8657 (1985).
Gu et al., "Independent Control of Immunoglobulin Switch Recombination at Individual Switch Regions Evidenced through Cre-loxP-Mediated Gene Targeting," Cell 73:1155-1164 (1993).
Hoess et al., "P1 site-specific recombination: Nucleotide sequence of the recombining sites," Proc. Natl. Acad. Sci. USA 79:3398-3402 (1982).
Graham et al., "A New Technique for the Assay of infectivity of Human Adenovirus 5 DNA," Virology 52:456-467 (1973).
Pavlakis et al., "Regulation of metallothionein-growth hormone hybrid gene in bovine papilloma virus," Proc. Natl. Acad. Sci. USA 80:397-401 (1983).
Gorman et al., "The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA-mediated transfection," Proc. Natl. Acad. Sci. USA 79:6777-6781 (1982).
Alvarado-Urbina et al., "Automated Synthesis of Gene Fragments" Science, 214:270-274 (1981).
Gu et al. Cell 73: 1155, 1993.

LandOfFree

Say what you really think

Search LandOfFree.com for the USA inventors and patents. Rate them and share your experience with other people.

Rating

Production of antibodies using Cre-mediated site-specific recomb does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Production of antibodies using Cre-mediated site-specific recomb, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Production of antibodies using Cre-mediated site-specific recomb will most certainly appreciate the feedback.

Rate now

     

Profile ID: LFUS-PAI-O-2038666

  Search
All data on this website is collected from public sources. Our data reflects the most accurate information available at the time of publication.