Claims
- 1. A genetically engineered cell in which a gene encoding an enzyme has been disrupted, wherein said gene encodes an enzyme selected from the group consisting of a Forssman glycolipid synthetase and a PK enzyme, wherein said PK enzyme is an enzyme associated with the synthesis of a PK carbohydrate.
- 2. The genetically engineered cell of claim 1, wherein both chromosomal copies of said gene have been disrupted.
- 3. The genetically engineered cell of claim 1, wherein said gene encoding an enzyme is a porcine gene.
- 4. The genetically engineered cell of claim 1, further comprising at least one additional gene that has been disrupted, wherein said at least one additional gene encodes a polypeptide comprising an antigenic determinant which is recognized by a desired recipient organism or said at least one gene encodes a protein associated with the synthesis of a molecule comprising an antigenic determinant recognized by the desired recipient organism.
- 5. The genetically engineered cell of claim 4, wherein said desired recipient organism is a human being.
- 6. The genetically engineered cell of claim 4, wherein a plurality of genes encoding polypeptides comprising antigenic determinants recognized by a desired recipient organism have been disrupted.
- 7. The genetically engineered cell of claim 4, 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 determninants recognized by the recipient organism have been disrupted.
- 8. The genetically engineered cell of claim 4, wherein substantially all of the genes encoding polypeptides comprising antigenic determinants recognized by the recipient organism have been disrupted.
- 9. The genetically engineered cell of claim 4, wherein said cell is from an organism selected from the group consisting of a mammal, a marsupial, a teleost fish, and an avian.
- 10. The genetically engineered cell of claim 9, wherein said mammal is selected from the group consisting of a non-human primate, a sheep, a goat, and a cow.
- 11. The genetically engineered cell of claim 9, wherein said avian is a chicken.
- 12. The genetically engineered cell of claim 9, wherein said cell is from a pig.
- 13. The genetically engineered cell of claim 12, 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.
- 14. The genetically engineered cell of claim 1, wherein said gene encoding an enzyme 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 of a nucleic acid selected from the group consisting of a nucleic acid encoding Forssman glycolipid synthetase, a nucleic acid encoding PK enzyme, and a portion of the foregoing nucleic acids.
- 15. The genetically engineered cell of claim 1, wherein said gene encoding an enzyme 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 of a nucleic acid selected from the group consisting of a nucleic acid encoding Forssman glycolipid synthetase, a nucleic acid encoding PK enzyme, and a portion of the foregoing nucleic acids.
- 16. The genetically engineered cell of claim 1, wherein said gene encoding an enzyme has been disrupted by replacing at least one chromosomal copy of said gene with a homologous sequence comprising a deletion in a nucleic acid selected from the group consisting of a nucleic acid encoding Forssman glycolipid synthetase, a nucleic acid encoding PK enzyme, and a portion of the foregoing nucleic acids.
- 17. The genetically engineered cell of claim 1, wherein said gene encoding an enzyme 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.
- 18. The genetically engineered cell of claim 17, wherein said replacement nucleotide sequence comprises a gene encoding a marker or a gene encoding a polypeptide from said desired recipient organism.
- 19. The genetically engineered cell of claim 4, wherein said at least one additional gene is a gene other than the GGTA1 gene.
- 20. The genetically engineered cell of claim 4, wherein said at least one additional 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 4, wherein said antigenic determinant comprises a polypeptide, a carbohydrate, or a lipid.
- 22. The genetically engineered cell of claim 4, wherein said gene encoding an enzyme is a gene other than a canine gene, a murine gene, or a human gene.
- 23. The genetically engineered cell of claim 1, wherein said gene encoding an enzyme comprises the sequence of SEQ ID NO: 29 or SEQ ID NO: 39.
- 24. The genetically engineered cell of claim 1, wherein said gene encoding an enzyme comprises a sequence encoding the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 40.
- 25. The genetically engineered cell of claim 1, wherein said gene encoding a Forssman glycolipid synthetase is selected from the group consisting of a gene comprising a sequence having at least 99% identity to the sequence of SEQ ID NO: 29, a gene comprising a sequence having at least 97% identity to the sequence of SEQ ID NO: 29, a gene comprising a sequence having at least 95% identity to the sequence of SEQ ID NO: 29, and a gene comprising a sequence having at least 90% identity to the sequence of SEQ ID NO: 29, wherein nucleotide sequence identity is determined using BLASTN version 2.0 with the default parameters.
- 26. The genetically engineered cell of claim 1, wherein said gene encoding a Forssman glycolipid synthetase has nucleic acid sequence identity to a gene encoding SEQ ID NO: 30, wherein the gene has nucleic acid sequence identity selected from the group consisting of 99% nucleic acid sequence identity, 97% nucleic acid sequence identity, 95% nucleic acid sequence identity, and 90% nucleic acid sequence identity to the sequence encoding the amino acid sequence in SEQ ID NO: 30.
- 27. The genetically engineered cell of claim 1, wherein said gene encoding a PK enzyme is selected from the group consisting of a gene comprising a sequence having at least 99% identity to the sequence of SEQ ID NO: 39, a gene comprising a sequence having at least 97% identity to the sequence of SEQ ID NO: 39, a gene comprising a sequence having at least 95% identity to the sequence of SEQ ID NO: 39, and a gene comprising a sequence having at least 90% identity to the sequence of SEQ ID NO: 39, wherein nucleotide sequence identity is determined using BLASTN version 2.0 with the default parameters.
- 28. The genetically engineered cell of claim 1, wherein said gene encoding a Forssman glycolipid synthetase has nucleic acid sequence identity to a gene encoding SEQ ID NO: 40, wherein the gene has nucleic acid sequence identity selected from the group consisting of 99% nucleic acid sequence identity, 97% nucleic acid sequence identity, 95% nucleic acid sequence identity, and 90% nucleic acid sequence identity to the sequence encoding the amino acid sequence in SEQ ID NO: 40.
- 29. The genetically engineered cell of claim 1, wherein said gene comprises at least 600, 700, 800, 900, 1000 or 1100 consecutive nucleotides of the sequence set forth in SEQ ID NO: 29 or SEQ ID NO: 39.
- 30. The genetically engineered cell of claim 1, wherein said gene encodes a polypeptide comprising at least 100, 150, 200, 250 or 290 consecutive amino acids of the sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 40.
- 31. A genetically engineered cell in which a gene comprising the sequence selected from the group consisting of SEQ ID NO: 29 and SEQ ID NO: 39 has been disrupted.
- 32. The genetically engineered cell of claim 31, wherein both chromosomal copies of said gene have been disrupted.
- 33. The genetically engineered cell of claim 32, further comprising at least one additional gene that has been disrupted, wherein said at least one additional gene encodes a polypeptide comprising an antigenic determinant which is recognized by a desired recipient organism or said at least one gene encodes a protein associated with the synthesis of a molecule comprising an antigenic determinant recognized by the desired recipient organism.
- 34. The genetically engineered cell of claim 33, wherein said desired recipient organism is a human being.
- 35. The genetically engineered cell of claim 33, wherein a plurality of genes encoding polypeptides comprising antigenic determinants recognized by a desired recipient organism have been disrupted.
- 36. The genetically engineered cell of claim 33, 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 more than 40 genes encoding polypeptides comprising antigenic determinants recognized by the recipient organism have been disrupted.
- 37. The genetically engineered cell of claim 33, wherein substantially all of the genes encoding polypeptides comprising antigenic determinants recognized by the recipient organism have been disrupted.
- 38. The genetically engineered cell of claim 1, wherein said gene encoding a PK enzyme is a gene encoding a porcine homolog of Galβ1-4Glcβ1-Cer α1,4-Galactosyltransferase.
- 39. A recombinant nucleic acid comprising:
a 5′ region homologous to a portion of a gene responsible for the production of an antigenic determinant, wherein said gene is selected from the group consisting of a Forssman glycolipid synthetase and a PK enzyme; a 3′ region homologous to a portion of said gene; and a nucleotide sequence which prevents the synthesis of the Forssman glycolipid synthetase or the PK enzyme, said nucleotide sequence being disposed between said 5′ region and said 3′ region.
- 40. The recombinant nucleic acid of claim 39, wherein said recombinant nucleic acid comprises a 5′ region homologous to a portion of a gene comprising a sequence selected from the group consisting of SEQ ID NO: 29 and SEQ ID NO: 39, and a 3′ region homologous to a portion of a gene comprising a sequence selected from the group consisting of SEQ ID NO: 29 and SEQ ID NO: 39.
- 41. The recombinant nucleic acid of claim 39, wherein said recombinant nucleic acid comprises a 5′ region homologous to a portion of a gene comprising a sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NO: 30 and SEQ ID NO: 40, and a 3′ region homologous to a portion of a gene comprising a sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NO: 29 and SEQ ID NO: 40.
- 42. The recombinant nucleic acid of claim 39, wherein at least a portion of said nucleotide sequence which prevents the synthesis of said Forssman glycolipid synthetase or said PK enzyme is disposed between said 5′ region and said 3′ region, said at least a portion containing an alteration therein which prevents the synthesis of said Forssman glycolipid synthetase or said PK enzyme.
- 43. The recombinant nucleic acid sequence of claim 42, wherein said alteration comprises at least one deletion in a nucleic acid selected from the group consisting of a nucleic acid encoding Forssman glycolipid synthetase, a nucleic acid encoding PK enzyme, and a portion of the foregoing nucleic acids.
- 44. The recombinant nucleic acid sequence of claim 42, wherein said alteration comprises a stop codon in the open reading frame of a nucleic acid selected from the group consisting of a nucleic acid encoding Forssman glycolipid synthetase, a nucleic acid encoding PK enzyme, and a portion of the foregoing nucleic acidsof said gene.
- 45. The recombinant nucleic acid sequence of claim 42, wherein said alteration comprises a nucleotide sequence containing a stop codon in all three reading frames of a nucleic acid selected from the group consisting of a nucleic acid encoding Forssman glycolipid synthetase, a nucleic acid encoding PK enzyme, and a portion of the foregoing nucleic acids.
- 46. The recombinant nucleic acid sequence of claim 42, wherein said alteration comprises a replacement sequence comprising a gene encoding a marker or a gene encoding a polypeptide from said desired recipient organism.
- 47. The recombinant nucleic acid sequence of claim 39, wherein said nucleotide sequence which prevents the synthesis of said Forssman glycolipid synthetase or said PK enzyme comprises a positive marker indicative of integration somewhere in the genome and a negative marker indicative of random integration in the genome.
- 48. The recombinant nucleic acid sequence of claim 47, wherein said positive marker is flanked by nucleotide sequences homologous to the genomic region in which integration via homologous recombination is desired.
- 49. The recombinant nucleic acid sequence of claim 39, wherein said nucleotide sequence which prevents the synthesis of said Forssman glycolipid synthetase or said PK enzyme 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 Forssman glycolipid synthetase or a PK enzyme if homologous recombination occurs.
- 50. The recombinant nucleic acid sequence of claim 39, wherein said nucleotide sequence which prevents the synthesis of said Forssman glycolipid synthetase or said PK enzyme 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.
- 51. The recombinant nucleic acid sequence of claim 39, further comprising at least one nucleic acid encoding a detectable polypeptide, said at least one nucleic acid being operably linked to a promoter.
- 52. The recombinant nucleic acid sequence of claim 51, 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.
- 53. The recombinant nucleic acid sequence of claim 51, wherein said detectable polypeptide is selected from the group consisting of CD8, green fluorescent protein (GFP), and Red fluorescent protein.
- 54. The recombinant nucleic acid sequence of claim 51, 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.
- 55. The recombinant nucleic acid sequence of claim 54, wherein said site which enables subsequent removal of a non-homologous sequence is a Lox P site or an Frt site.
- 56. The recombinant nucleic acid sequence of claim 51, further comprising at least one nucleic acid encoding a fusion polypeptide, said at least one nucleic acid being operably linked to a promoter, wherein said fusion polypeptide is selected from the group consisting of Flag tag, HA tag, c-myc, GST, mbp, and polyhistidine.
- 57. The recombinant nucleic acid of claim 39, wherein said gene responsible for the production of said Forssman glycolipid synthetase or said PK enzyme is a porcine gene.
- 58. The recombinant nucleic acid of claim 39, wherein said PK enzyme is Galβ1-4Glcβ1-Cer α1,4-Galactosyltransferase.
- 59. A method of disrupting a gene comprising a sequence selected from the group consisting of SEQ ID NO: 29 and SEQ ID NO: 39, 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.
- 60. The method of claim 57, 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.
- 61. The method of claim 58, wherein said double stranded break is introduced using at least one zinc finger endonuclease protein.
- 62. The method of claim 57, wherein said disruption in said coding region comprises at least one stop codon in one open reading frame of said gene.
- 63. The method of claim 60, wherein said disruption comprises a nucleotide sequence containing a stop codon in all three reading frames.
- 64. An isolated nucleic acid sequence comprising the sequence of SEQ ID NO: 29.
- 65. An isolated nucleic acid sequence comprising a nucleic acid sequence selected from the group consisting of a nucleic acid sequence comprising a sequence having at least 99% identity to the sequence of SEQ ID NO: 29, a nucleic acid sequence comprising a sequence having at least 97% identity to the sequence of SEQ ID NO: 29, a nucleic acid sequence comprising a sequence having at least 95% identity to the sequence of SEQ ID NO: 29, and a nucleic acid sequence comprising a sequence having at least 99% identity to the sequence of SEQ ID NO: 29, wherein nucleotide sequence identity is determined using BLASTN version 2.0 with the default parameters.
- 66. An isolated nucleic acid sequence comprising a sequence comprising at least 600, 700, 800, 900 or 1000 consecutive nucleotides of the sequence set forth in SEQ ID NO: 29.
- 67. An isolated nucleic acid encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 30.
- 68. An isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 30.
- 69. An isolated nucleic acid sequence comprising the sequence of SEQ ID NO: 39.
- 70. An isolated nucleic acid sequence comprising a nucleic acid sequence selected from the group consisting of a nucleic acid sequence comprising a sequence having at least 99% identity to the sequence of SEQ ID NO: 39, a nucleic acid sequence comprising a sequence having at least 97% identity to the sequence of SEQ ID NO: 39, a nucleic acid sequence comprising a sequence having at least 95% identity to the sequence of SEQ ID NO: 39, and a nucleic acid sequence comprising a sequence having at least 90% identity to the sequence of SEQ ID NO: 39, wherein nucleotide sequence identity is determined using BLASTN version 2.0 with the default parameters.
- 71. An isolated nucleic acid sequence comprising a sequence comprising at least 600, 700, 800, 900 or 1000 consecutive nucleotides of the sequence set forth in SEQ ID NO: 39.
- 72. An isolated nucleic acid encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 40.
- 73. An isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 40.
RELATED APPLICATIONS
[0001] This application is a continuation in part of U.S. patent application Ser. No. 10/147,286, entitled Tissues or Organs for Use in Xenotransplantation, filed May 14, 2002, the disclosure of which is incorporated herein by reference in its entirety. U.S. patent application Ser. No. 10/147,286 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 |
Continuation in Parts (1)
|
Number |
Date |
Country |
Parent |
10147286 |
May 2002 |
US |
Child |
10303686 |
Nov 2002 |
US |