What is claimed is:
1. An expression cassette comprising
(a) flanking regions which are homologous to a region of a ribosomal RNA gene from an organism selected from the group consisting of Leishmania spp., Crithidia spp. or Leptomonas spp.;
(b) intergenic regions which contain information required for RNA transcript processing in the organism; and
(c) a marker gene operably linked to the intergenic regions which allows selection of individuals of the organism which are transfected with the DNA molecule.
2. The expression cassette of claim 1, wherein the region of a ribosomal RNA gene is a conserved region of the small subunit of the ribosomal RNA gene of a Leshmania sp.
3. The expression cassette of claim 1, consisting essentially of the larger fragment resulting from a Swa1 digest of pIR1-SAT.
4. The expression cassette of claim 1, further comprising a second gene encoding a protein, wherein the second gene is operably linked to the intergenic regions.
5. The expression cassette of claim 4, wherein the second gene encodes a protein selected from the group consisting of a green fluorescent protein, insulin, -interferon, tissue plasminogen activator, -interferon, erythropoietin, Factor VIII, and a protein which is deficient or inactive in a lysosomal storage disease.
6. The expression cassette of claim 4, consisting essentially of the larger fragment resulting from a Swa1 digest of pIR1-SAT, and the second gene.
7. The expression cassette of claim 5, wherein the second gene encodes the green fluorescent protein.
8. An expression cassette comprising
(a) flanking regions which are homologous to a conserved region of the small subunit ribosomal RNA gene from an organism which undergoes trans-splicing;
(b) intergenic regions which contain information required for RNA transcript processing in the organism; and
(c) a marker gene operably linked to the intergenic regions which allows selection of individuals of the organism which are transfected with the DNA molecule.
9. The expression cassette of claim 8, wherein the organism is selected from the group consisting of Trypanosoma spp., Leishmania spp., Crithidia spp. and Leptomonas spp.
10. The expression cassette of claim 8, further comprising a second gene encoding a protein, wherein the second gene is operably linked to the intergenic regions.
11. The expression cassette of claim 10, wherein the organism is selected from the group consisting of Trypanosoma spp., Leishmania spp., Crithidia spp. or Leptomonas spp.
12. The expression cassette of claim 10, wherein the protein is selected from the group consisting of a green fluorescent protein, insulin, -interferon, tissue plasminogen activator, -interferon, erythropoietin, Factor VIII, and a protein which is deficient or inactive in a lysosomal storage disease.
13. The expression cassette of claim 12, wherein the protein is the green fluorescent protein.
14. An expression cassette comprising
(a) a promoter for a ribosomal RNA gene from an organism which undergoes trans-splicing;
(b) flanking sequences which are homologous to a chromosomal region of the organism;
(c) intergenic regions which contain information required for RNA transcript processing in the organism; and
(d) a marker gene operably linked to the intergenic regions which allows selection of individuals of the organism which are transfected with the DNA molecule.
15. The expression cassette of claim 14, wherein the organism is selected from the group consisting of Trypanosoma spp., Leishmania spp., Crithidia spp. and Leptomonas spp.
16. The expression cassette of claim 14, further comprising a second gene encoding a protein, wherein the second gene is operably linked to the intergenic regions.
17. The expression cassette of claim 16, wherein the protein is selected from the group consisting of a green fluorescent protein, insulin, -interferon, tissue plasminogen activator, -interferon, erythropoietin, Factor VIII, and a protein which is deficient or inactive in a lysosomal storage disease.
18. A recombinant plasmid comprising the expression cassette of claim 1 and DNA sequences which allow selection and replication of the vector in E. coli.
19. The recombinant plasmid of claim 18, consisting essentially of pIR1-SAT.
20. A recombinant plasmid comprising the expression cassette of claim 4 and DNA sequences which allow selection and replication of the vector in E. coli.
21. The recombinant plasmid of claim 20, wherein the second gene encodes a protein selected from the group consisting of a green fluorescent protein, insulin, -interferon, tissue plasminogen activator, -interferon, erythropoietin, Factor VIII, and a protein which is deficient or inactive in a lysosomal storage disease.
22. A recombinant plasmid comprising the expression cassette of claim 8 and DNA sequences which allow selection and replication of the vector in E. coli.
23. A recombinant plasmid comprising the expression cassette of claim 10 and DNA sequences which allow selection and replication of the vector in E. coli.
24. A recombinant plasmid comprising the expression cassette of claim 14 and DNA sequences which allow selection and replication of the vector in E. coli.
25. A recombinant plasmid comprising the expression cassette of claim 16 and DNA sequences which allow selection and replication of the vector in E. coli.
26. A host cell of an organism which undergoes trans-splicing transformed with the expression cassette of claim 4, wherein said host cell comprises a chromosome.
27. The host cell of claim 26, wherein the expression cassette is integrated into the chromosome.
28. The host cell of claim 27, wherein the organism is Leishmania tarentolae.
29. The host cell of claim 27, wherein the second gene encodes a protein selected from the group consisting of a green fluorescent protein, insulin, -interferon, tissue plasminogen activator, -interferon, erythropoietin, Factor VIII, and a protein which is deficient or inactive in a lysosomal storage disease.
30. The host cell of claim 27, wherein the second gene encodes a green fluorescent protein.
31. A host cell of an organism which undergoes trans-splicing transformed with the expression cassette of claim 10, wherein said host cell comprises a chromosome.
32. The host cell of claim 31, wherein the expression cassette is integrated into the chromosome.
33. The host cell of claim 32, wherein the organism is selected from the group consisting of Trypanosoma spp., Leishmania spp., Crithidia spp. and Leptomonas spp.
34. The host cell of claim 32, wherein the protein is selected from the group consisting of a green fluorescent protein, insulin, -interferon, tissue plasminogen activator, -interferon, erythropoietin, Factor VIII, and a protein which is deficient or inactive in a lysosomal storage disease.
35. A host cell of an organism which undergoes trans-splicing transformed with the expression cassette of claim 16, wherein said host cell comprises a chromosome.
36. The host cell of claim 35, wherein the expression cassette is integrated into the chromosome.
37. The host cell of claim 36, wherein the protein is selected from the group consisting of a green fluorescent protein, insulin, -interferon, tissue plasminogen activator, -interferon, erythropoietin, Factor VIII, and a protein which is deficient or inactive in a lysosomal storage disease.
38. A method of producing a protein, comprising:
(a) obtaining the host cell of claim 27, wherein the host cell further comprises cellular components, and
(b) culturing the host cell under conditions and for a time sufficient to produce the protein.
39. The method of claim 38, further comprising:
separating the protein from the cellular components.
40. The method of claim 38, wherein the protein is selected from the group consisting of a green fluorescent protein, insulin, -interferon, tissue plasminogen activator, -interferon, erythropoietin, Factor VIII, and a protein which is deficient or inactive in a lysosomal storage disease.
41. A method of producing a protein, comprising:
(a) obtaining the host cell of claim 32, wherein the host cell further comprises cellular components, and
(b) culturing the host cell under conditions and for a time sufficient to produce the protein.
42. The method of claim 41, further comprising:
separating the protein from the cellular components.
43. The method of claim 41, wherein the organism is selected from the group consisting of Trypanosoma spp., Leishmania spp., Crithidia spp. and Leptomonas spp.
44. The method of claim 41, wherein the protein is selected from the group consisting of a green fluorescent protein, insulin, -interferon, tissue plasminogen activator, -interferon, erythropoietin, Factor VIII, and a protein which is deficient or inactive in a lysosomal storage disease.
45. A method of producing a protein, comprising:
(a) obtaining the host cell of claim 36, wherein the host cell further comprises cellular components, and
(b) culturing the host cell under conditions and for a time sufficient to produce the protein.
46. The method of claim 45, further comprising:
separating the protein from the cellular components.
47. The method of claim 45, wherein the organism is selected from the group consisting of Trypanosoma spp., Leishmania spp., Crithidia spp. and Leptomonas spp.
48. The method of claim 45, wherein the protein is selected from the group consisting of a green fluorescent protein, insulin, -interferon, tissue plasminogen activator, -interferon, erythropoietin, Factor VIII, and a protein which is deficient or inactive in a lysosomal storage disease.
49. A method for studying virulence or pathogenicity in a trans-splicing organism, comprising infecting an experimental animal with the recombinant host cell of claim 27, wherein the protein is a green fluorescent protein.
50. A method for studying virulence or pathogenicity in a trans-splicing organism, comprising infecting an experimental animal with the recombinant host cell of claim 32, wherein the protein is a green fluorescent protein.
51. A method for studying virulence or pathogenicity in a trans-splicing organism, comprising infecting an experimental animal with the recombinant host cell of claim 36, wherein the protein is a green fluorescent protein.
52. A method of treating a disease or undesirable condition in a mammal, comprising infecting the mammal with an infectious strain of the host cell of claim 27, wherein the protein is useful for treating the disease or undesirable condition.
53. The method of claim 52, wherein the mammal is a human and the disease or undesirable condition is selected from the group consisting of osteoporosis, diabetes, cancer, severe anemia, short stature, hemophilia, and lysosomal storage diseases.
54. The method of claim 53, wherein the disease or undesirable condition is Goucher Disease or Fabry Disease.
55. A method of treating a disease or undesirable condition in a mammal, comprising infecting the mammal with an infectious strain of the host cell of claim 32, wherein the protein is useful for treating the disease or undesirable condition.
56. The method of claim 55, wherein the mammal is a human and the disease or undesirable condition is selected from the group consisting of osteoporosis, diabetes, cancer, severe anemia, short stature, hemophilia, and lysosomal storage diseases.
57. The method of claim 56, wherein the disease or undesirable condition is Goucher Disease or Fabry Disease.
58. A method of treating a disease or undesirable condition in a mammal, comprising infecting the mammal with an infectious strain of the host cell of claim 36, wherein the protein is useful for treating the disease or undesirable condition.
59. The method of claim 58, wherein the mammal is a human and the disease or undesirable condition is selected from the group consisting of osteoporosis, diabetes, cancer, severe anemia, short stature, hemophilia, and lysosomal storage diseases.
60. The method of claim 59, wherein the disease Goucher Disease or Fabry Disease.
61. A method of delivering a therapeutic protein to a desired site in a mammal, comprising
(a) selecting a trans-splicing organism which is capable of infecting the mammal and residing at the desired site;
(b) transfecting the trans-splicing organism with the expression cassette of claim 4, wherein the second gene encodes the therapeutic protein; and
(c) infecting the mammal with the transfected trans-splicing organism.
62. The method of claim 61, wherein the mammal is a human and the trans-splicing organism is selected from the group consisting of Leishmania spp. and Trypanosoma spp.
63. The method of claim 62, wherein the site is a lysosome and the trans-splicing organism is a Leishmania.
64. A method of delivering a therapeutic protein to a desired site in a mammal, comprising
(a) selecting a trans-splicing organism which is capable of infecting the mammal and residing at the desired site;
(b) transfecting the trans-splicing organism with the expression cassette of claim 10, wherein the second gene encodes the therapeutic protein; and
(c) infecting the mammal with the transfected trans-splicing organism.
65. The method of claim 64, wherein the mammal is a human and the trans-splicing organism is selected from the group consisting of Leishmania spp. and Trypanosoma spp.
66. The method of claim 65, wherein the site is a lysosome and the trans-splicing organism is a Leishmania.
67. A method of delivering a therapeutic protein to a desired site in a mammal, comprising
(a) selecting a trans-splicing organism which is capable of infecting the mammal and residing at the desired site;
(b) transfecting the trans-splicing organism with the expression cassette of claim 16, wherein the second gene encodes the therapeutic protein; and
(c) infecting the mammal with the transfected trans-splicing organism.
68. The method of claim 67, wherein the mammal is a human and the trans-splicing organism is selected from the group consisting of Leishmania spp. and Trypanosoma spp.
69. The method of claim 68, wherein the site is a lysosome and the trans-splicing organism is a Leishmania.
70. A kit for producing a recombinant protein, comprising the recombinant plasmid of claim 18, a living cell of the organism, and instructions.
71. The kit of claim 70, wherein the organism is Leishmania tarentolae.
72. A kit for producing a recombinant protein, comprising the recombinant plasmid of claim 22, a living cell of the organism, and instructions.
73. The kit of claim 72, wherein the organism is selected from the group consisting of Trypanosoma spp., Leishmania spp., Crithidia spp. and Leptomonas spp.
74. The kit of claim 73, wherein the recombinant plasmid is pIR1SAT.
75. The kit of claim 72, wherein the organism is selected from the group consisting of Crithidia spp., Leptomonas spp., and Leishmania tarentolae.
76. A kit for producing a recombinant protein, comprising the recombinant plasmid of claim 24, a living cell of the organism, and instructions.
77. The kit of claim 76, wherein the organism is selected from the group consisting of Trypanosoma spp., Leishmania spp., Crithidia spp. and Leptomonas spp.
78. The kit of claim 76, wherein the organism is selected from the group consisting of Crithidia spp., Leptomonas spp., and Leishmania tarentolae.
The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.
1. A method for reducing the number of layers in a multilayer signal routing device, the multilayer signal routing device having a plurality of electrically conductive signal path layers for routing electrical signals to and from at least one electronic component mounted on a surface of the multilayer signal routing device, the method comprising:
receiving electronic component information including an electrically conductive contact count characteristic, an electrically conductive contact pitch characteristic, an electrically conductive contact signal type characteristic, and an electrically conductive contact signal direction characteristic for at least one electronic component;
identifying an electronic component having a high density electrically conductive contact array package based at least in part upon at least one of the electrically conductive contact count characteristic and the electrically conductive contact pitch characteristic; and
routing electrical signals on the plurality of electrically conductive signal path layers in the multilayer signal routing device for connection to and from the high density electrically conductive contact array package based at least in part upon at least one of the electrically conductive contact signal type characteristic and the electrically conductive contact signal direction characteristic.
2. The method of claim 1, further comprising:
forming a plurality of electrically conductive vias in the multilayer signal routing device extending from the surface of the multilayer signal routing device to one of the plurality of electrically conductive signal path layers, the plurality of electrically conductive vias being arranged so as to form a channel in another of the plurality of electrically conductive signal path layers beneath the plurality of electrically conductive vias.
3. The method of claim 2, wherein the channel is configured to have one or more of a linear, circular, diamond, curved, stepped, staggered, and random shape, or a combination thereof.
4. The method of claim 2, wherein the channel is configured to be in one or more of a vertical, horizontal, diagonal, and random direction, or a combination thereof.
5. The method of claim 2, wherein the plurality of electrically conductive vias form at least a portion of an electrically conductive contact array for mating with the high density electrically conductive contact array package of the electronic component, and wherein at least a portion of the plurality of electrically conductive vias are positioned inside the electrically conductive contact array such that the channel is correspondingly formed inside the electrically conductive contact array.
6. The method of claim 2, wherein the multilayer signal routing device has an array of electrically conductive contacts formed on the surface thereof for mating with the high density electrically conductive contact array package of the electronic component, wherein at least a portion of the plurality of electrically conductive vias are formed outside the array of electrically conductive contacts, wherein each of the portion of the plurality of electrically conductive vias is electrically connected to peripheral ones of the electrically conductive contacts on the surface the multilayer signal routing device.
7. The method of claim 2, wherein the plurality of electrically conductive vias further extend from the surface of the multilayer signal routing device to different ones of the plurality of electrically conductive signal path layers.
8. The method of claim 2, wherein the plurality of electrically conductive vias form at least a portion of an electrically conductive contact array for mating with the high density electrically conductive contact array package of the electronic component, and wherein at least a portion of the plurality of electrically conductive vias are positioned within the electrically conductive contact array such that the channel extends across the electrically conductive contact array.
9. The method of claim 2, wherein the plurality of electrically conductive vias form at least a portion of an electrically conductive contact array for mating with the high density electrically conductive contact array package of the electronic component, wherein the electrically conductive contact array has one or more of a square, triangular, circular, and random electrically conductive contact pattern, or a combination thereof.
10. The method of claim 2, wherein at least two of the electrical signals are differential electrical signals, wherein the differential electrical signals are at least partially routed together in the channel formed in another of the plurality of electrically conductive signal path layers beneath the plurality of electrically conductive vias.
11. The method of claim 1, wherein the multilayer signal routing device has at least one electrically conductive power layer for providing power to the electronic component mounted on the surface of the multilayer signal routing device, the method further comprising:
forming a plurality of electrically conductive vias in the multilayer signal routing device extending from the surface of the multilayer signal routing device to at least one of the at least one electrically conductive power layer, each of the plurality of electrically conductive vias being electrically connected to at least one respective electrically conductive power contact on the surface of the multilayer signal routing device, each of the at least one electrically conductive power contact forming a portion of an electrically conductive contact array for mating with the high density electrically conductive contact array package of the electronic component;
wherein a channel is formed in each of the plurality of electrically conductive signal path layers beneath the electrically conductive power contacts.
12. The method of claim 11, wherein the channel is configured to have one or more of a linear, circular, diamond, curved, stepped, staggered, and random shape, or a combination thereof.
13. The method of claim 11, wherein the channel is configured to be in one or more of a vertical, horizontal, diagonal, and random direction, or a combination thereof.
14. The method of claim 11, wherein at least a portion of the electrically conductive power contacts are positioned inside the electrically conductive contact array such that the channel is correspondingly formed inside the electrically conductive contact array.
15. The method of claim 11, wherein at least a portion of the plurality of electrically conductive vias are formed outside the array of electrically conductive contacts, wherein each of the portion of the plurality of electrically conductive vias is electrically connected to at least one respective electrically conductive power contact that is located on the periphery of the array of electrically conductive contacts.
16. The method of claim 11, wherein at least a portion of the electrically conductive power contacts are positioned within the electrically conductive contact array such that the channel extends across the electrically conductive contact array.
17. The method of claim 11, wherein the electrically conductive contact array has one or more of a square, triangular, circular, and random electrically conductive contact pattern, or a combination thereof.
18. The method of claim 11, wherein at least two of the electrical signals are differential electrical signals, wherein the differential electrical signals are at least partially routed together in the channel formed in one of the plurality of electrically conductive signal path layers beneath the electrically conductive power contacts.
19. The method of claim 1, wherein the multilayer signal routing device has at least one electrically conductive ground layer for providing a ground reference to the electronic component mounted on the surface of the multilayer signal routing device, the method further comprising:
forming a plurality of electrically conductive vias in the multilayer signal routing device extending from the surface of the multilayer signal routing device to at least one of the at least one electrically conductive ground layer, each of the plurality of electrically conductive vias being electrically connected to at least one respective electrically conductive ground contact on the surface of the multilayer signal routing device, each of the at least one electrically conductive ground contact forming a portion of an electrically conductive contact array for mating with the high density electrically conductive contact array package of the electronic component;
wherein a channel is formed in each of the plurality of electrically conductive signal path layers beneath the electrically conductive ground contacts.
20. The method of claim 19, wherein the channel is configured to have one or more of a linear, circular, diamond, curved, stepped, staggered, and random shape, or a combination thereof.
21. The method of claim 19, wherein the channel is configured to be in one or more of a vertical, horizontal, diagonal, and random direction, or a combination thereof.
22. The method of claim 19, wherein at least a portion of the electrically conductive ground contacts are positioned inside the electrically conductive contact array such that the channel is correspondingly formed inside the electrically conductive contact array.
23. The method of claim 19, wherein at least a portion of the plurality of electrically conductive vias are formed outside the array of electrically conductive contacts, wherein each of the portion of the plurality of electrically conductive vias is electrically connected to at least one respective electrically conductive ground contact that is located on the periphery of the array of electrically conductive contacts.
24. The method of claim 19, wherein at least a portion of the electrically conductive ground contacts are positioned within the electrically conductive contact array such that the channel extends across the electrically conductive contact array.
25. The method of claim 19, wherein the electrically conductive contact array has one or more of a square, triangular, circular, and random electrically conductive contact pattern, or a combination thereof.
26. The method of claim 19, wherein at least two of the electrical signals are differential electrical signals, wherein the differential electrical signals are at least partially routed together in the channel formed in one of the plurality of electrically conductive signal path layers beneath the electrically conductive ground contacts.
27. The method of claim 1, wherein the multilayer signal routing device has at least one electrically conductive shared powerground layer for providing powerground to the electronic component mounted on the surface of the multilayer signal routing device, the method further comprising:
forming a plurality of electrically conductive vias in the multilayer signal routing device extending from the surface of the multilayer signal routing device to at least one of the at least one electrically conductive shared powerground layer, each of the plurality of electrically conductive vias being electrically connected to at least one respective electrically conductive powerground contact on the surface of the multilayer signal routing device, each of the at least one electrically conductive powerground contact forming at least a portion of an electrically conductive contact array for mating with the high density electrically conductive contact array package of the electronic component;
wherein a channel is formed in each of the plurality of electrically conductive signal path layers beneath each of the at least one electrically conductive powerground contact.
28. The method of claim 1, wherein the surface of the multilayer signal routing device is an inner surface of the multilayer signal routing device, and the at least one electronic component is mounted on the inner surface of the multilayer signal routing device.
29. The method of claim 28, wherein the at least one electronic component has at least one first electrically conductive contact formed on a first side thereof, wherein the at least one first electrically conductive contact is electrically connected to at least one first corresponding electrically conductive contact formed on the inner surface of the multilayer signal routing device.
30. The method of claim 29, wherein the at least one electronic component has at least one second electrically conductive contact formed on a second side thereof, wherein the at least one second electrically conductive contact is electrically connected to at least one second corresponding electrically conductive contact formed on another inner surface of the multilayer signal routing device.