Claims
- 1. A genetically engineered cell in which at least one gene encoding a polypeptide comprising an antigenic determinant which is recognized by a desired recipient organism or at least one gene which encodes a protein associated with the synthesis of a molecule comprising an antigenic determinant recognized by the desired recipient organism has been disrupted.
- 2. The genetically engineered cell of claim 1, wherein both chromosomal copies of said at least one gene have been disrupted.
- 3. The genetically engineered cell of claim 1, wherein at least one gene encoding a polypeptide comprising an antigenic determinant which is recognized by human beings has been disrupted.
- 4. The genetically engineered cell of claim 2, wherein a plurality of genes encoding polypeptides comprising antigenic determinants recognized by a desired recipient organism have been disrupted.
- 5. The genetically engineered cell of claim 2, wherein at least two, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 35, at least 40 or than 40 genes encoding polypeptides comprising antigenic determinants recognized by the recipient organism have been disrupted.
- 6. The genetically engineered cell of claim 2, wherein substantially all of the genes encoding polypeptides comprising antigenic determinants recognized by the recipient organism have been disrupted.
- 7. The genetically engineered cell of claim 2, wherein said cell is from an organism selected from the group consisting of a mammal, a marsupial, a teleost fish, and an avian.
- 8. The genetically engineered cell of claim 7, wherein said mammal is selected from the group consisting of a non-human primate, a sheep, a goat, and a cow.
- 9. The genetically engineered cell of claim 7, wherein said avian is a chicken.
- 10. The genetically engineered cell of claim 7, wherein said cell is from a pig.
- 11. The genetically engineered cell of claim 10, wherein said cell is selected from the group consisting of primary pig skin fibroblasts, pig granulosa cells, pig stem cells, pig germ cells, pig peripheral blood cells, pig hematopoetic stem cells and primary pig fetal fibroblasts.
- 12. The genetically engineered cell of claim 1, wherein said at least one gene has been disrupted by replacing at least one chromosomal copy of said gene with a homologous sequence comprising a stop codon in the open reading frame which encodes said polypeptide.
- 13. The genetically engineered cell of claim 1, wherein said at least one gene has been disrupted by replacing at least one chromosomal copy of said gene with a homologous sequence comprising a stop codon in all three reading frames.
- 14. The genetically engineered cell of claim 1, wherein said at least one gene has been disrupted by replacing at least one chromosomal copy of said gene with a homologous sequence comprising a deletion.
- 15. The genetically engineered cell of claim 1, wherein said at least one gene has been disrupted by replacing at least one chromosomal copy of said gene with a non-homologous replacement nucleotide sequence flanked by nucleotide sequences homologous to a genomic sequence in which homologous recombination is desired.
- 16. The genetically engineered cell of claim 15, wherein said replacement nucleotide sequence comprises a gene encoding a marker or a gene encoding a polypeptide from said desired recipient organism.
- 17. The genetically engineered cell of claim 16, wherein said gene encoding a polypeptide from said desired recipient organism comprises a gene encoding a major histocompatability complex (MHC) Protein.
- 18. The genetically engineered cell of claim 2, wherein said desired recipient organism is a human being.
- 19. The genetically engineered cell of claim 2, wherein said at least one gene is a gene other than the GGTA1 gene.
- 20. The genetically engineered cell of claim 2, wherein said gene encodes a polypeptide that includes an antigenic determinant or a polypeptide associated with the synthesis or modification of an antigenic determinant.
- 21. The genetically engineered cell of claim 1, wherein said antigenic determinant comprises a polypeptide, a carbohydrate, or a lipid.
- 22. A recombinant nucleic acid comprising a 5′ region homologous to a portion of a gene responsible for the production of an antigenic determinant recognized by a desired recipient organism or a 5′ region homologous to a portion of a gene encoding a polypeptide associated with the synthesis of a molecule comprising an antigenic determinant recognized by said desired organism, a 3′ region homologous to a portion of a gene responsible for the production of an antigenic determinant recognized by said desired recipient organism or a 3′ region homologous to a portion of a gene encoding a polypeptide associated with the synthesis of a molecule comprising an antigenic determinant recognized by said desired organism, and a nucleotide sequence which prevents the synthesis of an antigenic determinant recognized by said desired recipient organism, said nucleotide sequence being disposed between said 5′ region and said 3′ region.
- 23. The recombinant nucleic acid of claim 22, wherein at least a portion of said nucleotide sequence which prevents the synthesis of an antigenic determinant recognized by said desired recipient organism is disposed between said 5′ region and said 3′ region, said at least a portion containing an alteration therein which prevents the synthesis of an antigenic determinant recognized by said desired recipient organism.
- 24. The recombinant nucleic acid sequence of claim 22, wherein said alteration comprises at least one deletion.
- 25. The recombinant nucleic acid sequence of claim 22, wherein said alteration comprises a stop codon in the open reading frame which encodes a polypeptide comprising an antigenic determinant recognized by said desired recipient organism.
- 26. The recombinant nucleic acid sequence of claim 22, wherein said alteration comprises a nucleotide sequence containing a stop codon in all three reading frames.
- 27. The recombinant nucleic acid sequence of claim 22, wherein said alteration comprises a replacement sequence comprising a gene encoding a marker or a gene encoding a polypeptide from said desired recipient organism.
- 28. The recombinant nucleic acid sequence of claim 27, wherein said gene encoding a polypeptide from said desired recipient organism comprises a gene encoding an MHC Protein.
- 29. The recombinant nucleic acid sequence of claim 22, wherein said nucleotide sequence which prevents the synthesis of an antigenic determinant recognized by said desired recipient organism comprises a positive marker indicative of integration somewhere in the genome and a negative marker indicative of random integration in the genome.
- 30. The recombinant nucleic acid sequence of claim 29, wherein said positive marker is flanked by nucleotide sequences homologous to the genomic region in which integration via homologous recombination is desired.
- 31. The recombinant nucleic acid sequence of claim 22, wherein said nucleotide sequence which prevents the synthesis of an antigenic determinant recognized by said desired recipient organism comprises a promoterless marker gene flanked by nucleotide sequences which will put said marker gene under the control of the promoter which directs transcription of said gene encoding a polypeptide comprising an antigenic determent recognized by a desired recipient organism if homologous recombination occurs.
- 32. The recombinant nucleic acid sequence of claim 22, wherein said nucleotide sequence which prevents the synthesis of an antigenic determinant recognized by said desired recipient organism comprises a portion of a gene encoding a nonfunctional portion of a marker protein, said portion of said gene encoding a nonfunctional portion of a marker protein being flanked by nucleotide sequences homologous to the desired integration site.
- 33. The recombinant nucleic acid sequence of claim 22, further comprising at least one nucleic acid encoding a detectable polypeptide, said at least one nucleic acid being operably linked to a promoter.
- 34. The recombinant nucleic acid sequence of claim 33, wherein said recombinant nucleic acid comprises a nucleic acid encoding CD8 operably linked to a promoter and a nucleic acid encoding green fluorescent protein operably linked to a promoter.
- 35. The recombinant nucleic acid sequence of claim 33, wherein said detectable polypeptide is selected from the group consisting of CD8, green fluorescent protein (GFP), Red fluorescent protein, Flag tag, HA tag, c-myc, GST, mbp, and polyhistidine.
- 36. The recombinant nucleic acid sequence of claim 33, wherein at least one nucleic acid encoding a detectable polypeptide is flanked by a site which enables excision of said nucleic acid encoding a detectable polypeptide.
- 37. The recombinant nucleic acid sequence of claim 33, wherein said site which enables subsequent removal of a non-homologous sequence is a Lox P site or an Frt site.
- 38. The recombinant nucleic acid sequence of claim 22, wherein said gene responsible for the production of an antigenic determinant is a gene other than the GGTA1 gene.
- 39. The recombinant nucleic acid sequence of claim 22, wherein said gene is responsible for the production of an antigenic determinant which may be a polypeptide, a carbohydrate or a lipid, or which results from the modification of a polypeptide, carbohydrate or lipid.
- 40. A method of disrupting a gene encodes a polypeptide responsible for the production of an antigenic determinant recognized by a desired recipient organism comprising:
introducing a nucleic acid comprising a sequence homologous to at least a portion of the coding region of said gene into a cell, wherein said homologous sequence comprises a disruption in said coding region which prevents said cell from expressing the full length polypeptide normally encoded by said coding region; and replacing at least one chromosomal copy of said gene with said homologous sequence comprising said disruption in said coding region.
- 41. The method of claim 40, further comprising enhancing the rate of recombination by introducing a double stranded break in the nucleic acid in a region in the vicinity of the gene encoding a polypeptide comprising the antigenic determinant.
- 42. The method of claim 41, wherein said double stranded break is introduced using at least one zinc finger endonuclease domain.
- 43. The method of claim 40, wherein said disruption in said coding region comprises at least one stop codon in one open reading frame encoding said polypeptide.
- 44. The method of claim 43, wherein said disruption comprises a nucleotide sequence containing a stop codon in all three reading frames.
- 45. The method of claim 40, wherein said gene which encodes a polypeptide comprising an antigenic determinant recognized by a desired recipient organism is a gene other than the GGTA1 gene.
- 46. A method of identifying an antigenic determinant from a donor organism which is recognized by a recipient organism comprising:
obtaining a screening composition comprising a plurality of molecules from said donor organism; contacting said plurality of molecules with naturally occurring immunoglobulin family proteins; and identifying an antigenic determinant that is detected by said naturally occurring immunoglobulin family proteins.
- 47. The method of claim 46, wherein said screening composition comprises a plurality of molecules isolated from the surface of cells from said donor organism.
- 48. The method of claim 46, wherein said molecule is selected from the group consisting of a polypeptide, a lipid, a carbohydrate, and a molecule comprising any combination of the foregoing molecules.
- 49. The method of claim 46, wherein said naturally occurring immunoglobulin family proteins comprise immune sera from said recipient organism.
- 50. The method of claim 45, wherein said naturally occurring immunoglobulin family proteins comprise a polyclonal immunoglobulin population derived from said recipient organism.
- 51. A method of identifying a gene responsible for the production of an antigenic determinant from a donor organism that is recognized by a recipient organism comprising:
obtaining a plurality of nucleic acids encoding a plurality polypeptides from said donor organism and expressing the plurality of polypeptides; contacting said plurality of polypeptides with naturally occurring immunoglobulin family proteins present on the surface of or obtained from natural killer cells or T cells from the recipient organism; and identifying cells recognized by said naturally occurring immunoglobulin family proteins, whereby said cells comprise a gene from a donor organism which encodes a polypeptide comprising an antigenic determinant recognized by said recipient organism or a gene from said donor organism which encodes a polypeptide associated with the synthesis of a molecule comprising an antigenic determinant recognized by said recipient organism.
- 52. A method of identifying a gene from a donor organism which encodes a polypeptide comprising an antigenic determinant recognized by a recipient organism or a gene from said donor organism which encodes a polypeptide associated with the synthesis of a molecule comprising an antigenic determinant recognized by said recipient organism comprising:
obtaining a cDNA library comprising a plurality of genes encoding polypeptides from said donor organism; expressing said polypeptides in host cells; contacting said host cells with naturally occurring immunoglobulin family proteins which detect antigenic determinants recognized by said recipient organism; and identifying a host cell which expresses a polypeptide recognized by said naturally occurring Immunoglobulin family proteins.
- 53. The method of claim 52, wherein said naturally occurring immunoglobulin family proteins derived from said recipient organism comprises immune sera, wherein said expressed polypeptide is recognized an antibody in said immune sera.
- 54. The method of claim 52, wherein said naturally occurring immunoglobulin family proteins derived from said recipient organism comprise a polyclonal antibody population.
- 55. The method of claim 52, wherein said naturally occurring immunoglobulin family proteins comprise molecules present on the surface of or obtained from naturaly killer cells or T cells from the recipient organism.
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application Serial No. 60/291,394, Filed May 14, 2001, U.S. Provisional Application Serial No. 60/312,125, Filed Aug. 13, 2001, and U.S. Provisional Application Serial No. 60/367090, Filed Mar. 21, 2002; each of which is entitled TISSUES OR ORGANS FOR USE IN XENOTRANSPLANTATION. The disclosures of each of the foregoing Provisional Applications are incorporated by reference herein in their entireties.
Provisional Applications (3)
|
Number |
Date |
Country |
|
60291394 |
May 2001 |
US |
|
60312125 |
Aug 2001 |
US |
|
60367090 |
Mar 2002 |
US |