A method for engineering and utilizing large DNA vectors to target, via homologous recombination, and modify, in any desirable fashion, endogenous genes and chromosomal loci in eukaryotic cells. These large DNA targeting vectors for eukaryotic cells, termed LTVECs, are derived from fragments of clon
A method for engineering and utilizing large DNA vectors to target, via homologous recombination, and modify, in any desirable fashion, endogenous genes and chromosomal loci in eukaryotic cells. These large DNA targeting vectors for eukaryotic cells, termed LTVECs, are derived from fragments of cloned genomic DNA larger than those typically used by other approaches intended to perform homologous targeting in eukaryotic cells. Also provided is a rapid and convenient method of detecting eukaryotic cells in which the LTVEC has correctly targeted and modified the desired endogenous gene(s) or chromosomal locus (loci) as well as the use of these cells to generate organisms bearing the genetic modification.
대표청구항▼
1. A method of producing an antibody having a human immunoglobulin heavy chain variable region and a human heavy chain constant region, the method comprising: (a) providing a transgenic mouse whose genome comprises in its germline human unrearranged immunoglobulin heavy chain V, D, and J gene segmen
1. A method of producing an antibody having a human immunoglobulin heavy chain variable region and a human heavy chain constant region, the method comprising: (a) providing a transgenic mouse whose genome comprises in its germline human unrearranged immunoglobulin heavy chain V, D, and J gene segments, wherein the human unrearranged heavy chain V, D, and J gene segments in situ replace mouse endogenous immunoglobulin heavy chain V, D, and J gene segments, and the human unrearranged immunoglobulin heavy chain V, D, and J gene segments are operably linked to an endogenous mouse heavy chain constant region gene, wherein the mouse heavy chain constant region gene is located at an endogenous mouse immunoglobulin heavy chain constant region locus, wherein rearrangement of the human heavy chain V, D, and J gene segments in the mouse results in a rearranged human heavy chain variable region gene linked to the mouse heavy chain constant region gene, wherein the mouse in response to the antigen produces a hybrid antibody that comprises a human heavy chain variable region encoded by the rearranged human heavy chain variable region gene and a mouse heavy chain constant region encoded by the mouse heavy chain constant region gene, and wherein the mouse does not produce fully human antibodies;(b) stimulating an immune response in the mouse by exposing the mouse to an antigen;(c) preparing a hybridoma expressing the hybrid antibody from the spleen of the mouse stimulated with the antigen in step (b);(d) isolating DNA encoding the human heavy chain variable region of the hybrid antibody from the hydridoma of step (c);(e) operably linking the DNA encoding the human heavy chain variable region of the hybrid antibody to DNA encoding a human heavy chain constant region in a cell;(f) growing the cell under conditions such that the cell expresses an antibody comprising the human heavy chain variable region and the human heavy chain constant region; and(g) recovering the antibody. 2. The method of claim 1, wherein the mouse constant region gene consists of a murine Fc region. 3. A method of producing an antibody having a human immunoglobulin heavy chain variable region and a human heavy chain constant region, the method comprising: (a) providing a transgenic mouse whose genome comprises in its germline human unrearranged immunoglobulin heavy chain V, D, and J gene segments, wherein the human unrearranged heavy chain V, D, and J gene segments in situ replace mouse endogenous immunoglobulin heavy chain V, D, and J gene segments, and the human unrearranged immunoglobulin heavy chain V, D, and J gene segments are operably linked to an endogenous mouse heavy chain constant region gene, wherein the mouse heavy chain constant region gene is located at an endogenous mouse immunoglobulin heavy chain constant region locus, wherein rearrangement of the human heavy chain V, D, and J gene segments in the mouse results in a rearranged human heavy chain gene linked to the mouse heavy chain constant region gene, wherein the mouse in response to an antigen produces a hybrid antibody that comprises a human heavy chain variable region encoded by the rearranged human heavy chain variable region gene and a mouse heavy chain constant region encoded by the mouse heavy chain constant region gene, and wherein the mouse does not produce fully human antibodies;(b) stimulating an immune response in the mouse by exposing the mouse to an antigen;(c) isolating DNA encoding the human heavy chain variable region of the hybrid antibody from the mouse stimulated with the antigen in step (b);(d) operably linking the DNA encoding the human heavy chain variable region of the hybrid antibody to DNA encoding a human heavy chain constant region in a cell;(e) growing the cell under conditions such that the cell expresses an antibody comprising the human heavy chain variable region and the human heavy chain constant region; and(f) recovering the antibody. 4. The method of claim 3, wherein the mouse heavy chain constant region gene consists of a murine Fc region. 5. The method of claim 3, wherein the DNA encoding the human heavy chain variable region of the antibody is isolated in step (c) by PCR amplification or cDNA cloning. 6. The method of claim 1, wherein the cell is a CHO cell. 7. The method of claim 1, wherein the DNA encoding the human heavy chain variable region of the antibody is isolated in step (d) by PCR amplification or cDNA cloning.
연구과제 타임라인
LOADING...
LOADING...
LOADING...
LOADING...
LOADING...
이 특허에 인용된 특허 (33)
Berns Anton,NLX ; Robanus Maandag Els,NLX ; te Riele Hein,NLX, Gene targeting in animal cells using isogenic DNA constructs.
Murphy, Andrew J.; Yancopoulos, George D.; Karow, Margaret; Macdonald, Lynn; Stevens, Sean; Economides, Aris N.; Valenzuela, David M., Hybrid antibodies comprising human variable regions and mouse constant regions produced in a genetically modified mouse.
Economides, Aris N.; Murphy, Andrew J.; Valenzuela, David M.; Frendewey, David; Yancopoulos, George D., Method for genetically modifying mouse embryonic stem cell by homologous recombination.
Bradley Allan (Houston TX) Davis Ann (Houston TX) Hasty Paul (Houston TX), Methods for the genetic modification of endogenous genes in animal cells by homologous recombination.
Cabilly Shmuel ; Heyneker Herbert L. ; Holmes William E. ; Riggs Arthur D. ; Wetzel Ronald B., Methods of producing immunoglobulins, vectors and transformed host cells for use therein.
Le Mouellic, Herve; Brulet, Philippe, Procedure for specific replacement of a copy of a gene present in the recipient genome by the integration of a gene different from that where the integration is made.
Le Mouellic, Herve; Brulet, Philippe, Procedure for specific replacement of a copy of a gene present in the recipient genome by the integration of a gene different from that where the integration is made.
Surani Azim M. (Cambridge GB3) Neuberger Michael S. (Cambridge GB3) Bruggemann Marianne (Cambridge GB3), Production of antibodies from transgenic animals.
Barnes Wayne M. (223 Renaldo Dr. Chesterfield MO 63017), Thermostable DNA polymerase with enhanced thermostability and enhanced length and efficiency of primer extension.
Green, Larry L.; Ivanov, Vladimir E.; Davis, C. Geoffrey, Transgenic animals for producing specific isotypes of human antibodies via non-cognate switch regions.
※ AI-Helper는 부적절한 답변을 할 수 있습니다.