Claims for Patent: 6,270,989

Summary for Patent: 6,270,989

Title:	Protein production and delivery
Abstract:	The invention relates to novel human DNA sequences, targeting constructs, and methods for producing novel genes encoding thrombopoietin, DNase I, and .beta.-interferon by homologous recombination. The targeting constructs comprise at least: a) a targeting sequence; b) a regulatory sequence; c) an exon; and d) a splice-donor site. The targeting constructs, which can undergo homologous recombination with endogenous cellular sequences to generate a novel gene, are introduced into cells to produce homologously recombinant cells. The homologously recombinant cells are then maintained under conditions which will permit transcription of the novel gene and translation of the mRNA produced, resulting in production of either thrombopoietin, DNase I, or .beta.-interferon. The invention further relates to a methods of producing pharmaceutically useful preparations containing thrombopoietin, DNase I, or .beta.-interferon from homologously recombinant cells and methods of gene therapy comprising administering homologously recombinant cells producing thrombopoietin, DNase I, or .beta.-interferon to a patient for therapeutic purposes.
Inventor(s):	Treco; Douglas A. (Arlington, MA), Heartlein; Michael W. (Boxborough, MA), Hauge; Brian M. (Beverly, MA), Selden; Richard F (Wellesley, MA)
Assignee:	Transkaryotic Therapies, Inc. (Cambridge, MA)
Application Number:	08/406,030
Patent Claims:	1. A DNA construct which alters the expression of a targeted gene in a cell when the DNA construct is homologously recombined with a target site within the chromosomal DNA of the cell, the DNA construct comprising: (a) a targeting sequence homologous to the target site; (b) an exogenous regulatory sequence; (c) an exon; and (d) an unpaired splice-donor site at the 3' end of the exon; wherein following homologous recombination of the targeting sequence with the target site, the chromosomal DNA of the cell comprises the construct-derived exon in addition to all endogenous exons of the targeted gene. 2. The DNA construct of claim 1, wherein the DNA construct is linear. 3. The DNA construct of claim 1, wherein the exon sequence does not overlap with the targeting sequence. 4. The DNA construct of claim 1, wherein (a), (b), (c), and (d) are oriented such that, upon homologous recombination of the targeting sequence with the target site, the exogenous regulatory sequence controls expression of the targeted gene to produce a transcript comprising RNA corresponding to the construct-derived exon, construct-derived splice-donor site, and coding sequence of the targeted gene, wherein the RNA corresponding to the construct-derived splice-donor site of the transcript directs splicing to a splice-acceptor site in the transcript which corresponds to a site within the targeted gene. 5. The DNA construct of claim 4, wherein the splice-acceptor site of the transcript corresponds to the splice-acceptor site of the second exon of the targeted gene. 6. A DNA construct which alters the expression of a targeted gene in a cell when the DNA construct is homologously recombined with a target site within the chromosomal DNA of the cell, the DNA construct comprising: (a) a targeting sequence homologous to the target site; (b) an exogenous regulatory sequence; (c) an exon; and (d) an unpaired splice-donor site at the 3' end of the exon, wherein following homologous recombination of the targeting sequence with the target site, (b)-(d) are positioned upstream of the endogenous transcription initiation site of the targeted gene. 7. The DNA construct of claim 6, wherein (a), (b), (c), and (d) are oriented such that, upon homologous recombination of the targeting sequence with the target site, the exogenous regulatory sequence controls expression of the targeted gene to produce a transcript comprising RNA corresponding to the construct-derived exon, construct-derived splice-donor site, and coding sequence of the targeted gene, wherein the construct-derived splice-donor site of the transcript directs splicing to a splice-acceptor site in the transcript which corresponds to a site within the targeted gene. 8. The DNA construct of claim 7, wherein the splice-acceptor site of the transcript corresponds to the splice-acceptor site of the second exon of the targeted gene. 9. The DNA construct of claim 8, wherein the exon of the construct comprises coding sequence which, upon splicing of the construct-derived splice-donor site with the endogenous splice-acceptor site, is in-frame with coding sequence of the targeted gene. 10. The DNA construct of claim 9, wherein the exon of the construct comprises coding sequence that is the same as the coding sequence of the first exon of the targeted gene. 11. The DNA construct of claim 9, wherein the coding sequence of the exon of the construct is different from the coding sequence of the first exon of the targeted gene. 12. The DNA construct of claim 6, wherein the exon sequence does not overlap with the targeting sequence. 13. The DNA construct of claim 6, wherein the exon comprises a CAP site. 14. The DNA construct of claim 13, wherein the exon further comprises the nucleotide sequence ATG. 15. The DNA construct of claim 6, wherein the target site is upstream of an endogenous regulatory sequence of the targeted gene. 16. The DNA construct of claim 15, wherein the construct further comprises a second targeting sequence homologous to a sequence upstream of an endogenous regulatory sequence of the targeted gene. 17. The DNA construct of claim 6, wherein the targeted gene encodes a therapeutic protein. 18. The DNA construct of claim 6, wherein the targeted gene encodes a hormone, a cytokine, an antigen, an antibody, an enzyme, a clotting factor, a transport protein, a receptor, a regulatory protein, a structural protein, or a transcription factor. 19. The DNA construct of claim 6, wherein the targeted gene encodes a protein selected from the group consisting of calcitonin, insulin, insulinotropin, insulin-like growth factors, parathyroid hormone, nerve growth factors, TGF-.beta., tumor necrosis factor, glucagon, bone growth factor-2, bone growth factor-7, TSH-.beta., interleukin 1, interleukin 2, interleukin 3, interleukin 6, interleukin 11, interleukin 12, CSF-macrophage, immunoglobulins, catalytic antibodies, protein kinase C, superoxide dismutase, tissue plasminogen activator, urokinase, antithrombin III, DNase, tyrosine hydroxylase, blood clotting factor V, blood clotting factor VII, blood clotting factor VIII, blood clotting factor X, blood clotting factor XIII, apolipoprotein E, apolipoprotein A-I, globins, low density lipoprotein receptor, IL-2 receptor, IL-2 receptor antagonists, alpha-1 antitrypsin, immune response modifiers, and soluble CD4. 20. The DNA construct of claim 6, wherein the targeted gene encodes growth hormone. 21. The DNA construct of claim 6, wherein the targeted gene encodes blood clotting factor IX. 22. The DNA construct of claim 6, wherein the targeted gene encodes .alpha.-galactosidase. 23. The DNA construct of claim 6, wherein the targeted gene encodes glucocerebrosidase. 24. The DNA construct of claim 6, wherein the targeted gene encodes erythropoietin. 25. The DNA construct of claim 24, wherein the erythropoietin is human erythropoietin. 26. The DNA construct of claim 24, wherein the exon comprises coding sequence which is the same as the coding sequence of the first exon of the erythropoietin gene. 27. The DNA construct of claim 24, wherein the exon comprises coding sequence which is different from the coding sequence of the first exon of the erythropoietin gene. 28. The DNA construct of claim 24, wherein the exon comprises coding sequence which is the same as the coding sequence of the first exon of a growth hormone gene. 29. The DNA construct of claim 28, wherein the growth hormone gene encodes human growth hormone (hGH). 30. The DNA construct of claim 6, wherein the exogenous regulatory sequence is a promoter, an enhancer, a scaffold-attachment region, or a transcription factor binding site. 31. The DNA construct of claim 30, further comprising a second exogenous regulatory sequence. 32. The DNA construct of claim 30, wherein the exogenous regulatory sequence is a regulatory sequence of an adenovirus gene, a regulatory sequence of an SV-40 gene or a regulatory sequence of a cytomegalovirus gene. 33. The DNA construct of claim 30, wherein the exogenous regulatory sequence is a regulatory sequence of a mouse metallothionein-I gene, a regulatory sequence of a collagen gene, a regulatory sequence of an actin gene, a regulatory sequence of an immunoglobulin gene, a regulatory sequence of an HMG-CoA reductase gene, or a regulatory sequence of an EF-1.alpha. gene. 34. The DNA construct of claim 6, further comprising a gene. 35. The DNA construct of claim 6, further comprising one or more selectable marker genes. 36. The DNA construct of claim 6, further comprising an amplifiable marker gene. 37. A method of altering the expression of a targeted gene in a cell comprising the steps of: (a) providing a DNA construct comprising: (i) a targeting sequence; (ii) an exogenous regulatory sequence; (iii) an exon; and (iv) an unpaired splice-donor site at the 3' end of the exon, (b) providing a cell, the genome of which comprises (i) a target site homologous to the targeting sequence, and (ii) a targeted gene having an endogenous regulatory region; (c) transfecting the cell with the DNA construct, thereby producing a transfected cell; (d) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell the genome of which comprises the exogenous regulatory sequence, the construct-derived exon, and the construct-derived splice-donor site, in addition to all endogenous exons of the targeted gene; and (e) maintaining the homologously recombinant cell under conditions appropriate for transcription under the control of the exogenous regulatory sequence, to produce a transcript of the construct-derived exon, the targeted gene, and any sequence lying between the construct-derived exon and the targeted gene, wherein the RNA of the transcript corresponding to the construct-derived splice-donor site directs splicing to a splice-acceptor site in the transcript which corresponds to a site within the targeted gene. 38. The method of claim 37, wherein the DNA construct is linear. 39. The method of claim 37, wherein the exon sequence does not overlap with the targeting sequence. 40. The method of claim 37, wherein the splice-acceptor site of the transcript corresponds to the splice-acceptor site of the second endogenous exon of the targeted gene. 41. The method of claim 37, further comprising the steps of: (f) maintaining the homologously recombinant cell under conditions appropriate for splicing and translation of the transcript; and (g) confirming that a translation product of the transcript was produced. 42. A method of altering the expression of a gene in a cell comprising the steps of: (a) providing a cell, the genome of which comprises (i) a targeted gene having an endogenous regulatory region; (ii) a target site; (b) providing a DNA construct comprising: (i) a targeting sequence homologous to the target site; (ii) an exogenous regulatory sequence; (iii) an exon; and (iv) an unpaired splice-donor site at the 3' end of the exon, (c) transfecting the cell with the DNA construct, thereby producing a transfected cell; (d) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell the genome of which contains the exogenous regulatory sequence, the construct-derived exon, and the construct-derived splice-donor site, all upstream of the endogenous transcription initiation site of the targeted gene; and (e) maintaining the homologously recombinant cell under conditions appropriate for transcription under the control of the exogenous regulatory sequence, to produce a transcript of the construct-derived exon, the targeted gene, and any sequence lying between the construct-derived exon and the targeted gene. 43. The method of claim 42, wherein the splice-acceptor site of the transcript corresponds to the splice-acceptor site of the second exon of the targeted gene. 44. The method of claim 42, wherein the exon comprises a CAP site. 45. The method of claim 44, wherein the exon further comprises the nucleotide sequence ATG. 46. The method of claim 42, wherein the target site is upstream of an endogenous regulatory region of the targeted gene. 47. The method of claim 42, wherein the DNA construct further comprises a second targeting sequence homologous to a sequence upstream of an endogenous regulatory sequence of the targeted gene. 48. The method of claim 42, wherein the cell is a human cell. 49. The method of claim 42, wherein the targeted gene encodes a therapeutic protein. 50. The method of claim 42, wherein the targeted gene encodes a hormone, a cytokine, an antigen, an antibody, an enzyme, a clotting factor, a transport protein, a receptor, a regulatory protein, a structural protein, or a transcription factor. 51. The method of claim 42, wherein the targeted gene encodes a protein selected from the group consisting of calcitonin, insulin, insulinotropin, insulin-like growth factors, parathyroid hormone, nerve growth factors, TGF-.beta., tumor necrosis factor, glucagon, bone growth factor-2, bone growth factor-7, TSH-.beta., interleukin 1, interleukin 2, interleukin 3, interleukin 6, interleukin 11, interleukin 12, CSF-macrophage, immunoglobulins, catalytic antibodies, protein kinase C, superoxide dismutase, tissue plasminogen activator, urokinase, antithrombin III, DNase, tyrosine hydroxylase, blood clotting factor V, blood clotting factor VII, blood clotting factor VIII, blood clotting factor X, blood clotting factor XIII, apolipoprotein E, apolipoprotein A-I, globins, low density lipoprotein receptor, IL-2 receptor, IL-2 receptor antagonists, alpha-1 antitrypsin, immune response modifiers, and soluble CD4. 52. The method of claim 42, wherein the targeted gene encodes growth hormone. 53. The method of claim 42, wherein the targeted gene encodes blood clotting factor IX. 54. The method of claim 42, wherein the targeted gene encodes .alpha.-galactosidase. 55. The method of claim 42, wherein the targeted gene encodes glucocerebrosidase. 56. The method of claim 42, wherein the targeted gene encodes erythropoietin. 57. The method of claim 56, wherein the erythropoietin is human erythropoietin. 58. The method of claim 56, wherein the construct-derived exon comprises coding sequence which is the same as the coding sequence of the first exon of erythropoietin. 59. The method of claim 56, wherein the construct-derived exon comprises coding sequence which is different from the coding sequence of the first exon of erythropoietin. 60. The method of claim 59, wherein the construct-derived exon comprises coding sequence that is the same as the coding sequence of the first exon of human growth hormone. 61. The method of claim 60, wherein the growth hormone is human growth hormone. 62. The method of claim 42, wherein the exogenous regulatory sequence is a promoter, an enhancer, a scaffold-attachment region, or a transcription factor binding site. 63. The method of claim 62, wherein the DNA construct further comprises a second regulatory sequence. 64. The method of claim 62, wherein the exogenous regulatory sequence is a regulatory sequence of an adenovirus gene, a regulatory sequence of an SV-40 gene, or a regulatory sequence of a cytomegalovirus gene. 65. The method of claim 62, wherein the exogenous regulatory sequence is a regulatory sequence of a mouse metallothionein-I gene, a regulatory sequence of a collagen gene, a regulatory sequence of an actin gene, a regulatory sequence of an immunoglobulin gene, a regulatory sequence of an HMG-CoA reductase gene, or a regulatory sequence of an EF-1.alpha. gene. 66. The method of claim 42 further comprising the steps of: (f) maintaining the homologously recombinant cell under conditions appropriate for splicing and translation of the transcript; and (g) confirming that a translation product of the transcript was produced. 67. The method of claim 66 in which the targeted gene encodes erythropoietin. 68. A cultured vertebrate cell the genome of which bears a transcription unit comprising an exogenous regulatory sequence, an exogenous exon, and a splice-donor site at the 3' end of the exogenous exon, the splice-donor site being operatively linked to the endogenous splice-acceptor site of the second endogenous exon of an endogenous gene, wherein the genome comprises the exogenous exon in addition to all endogenous exons of the endogenous gene. 69. The cell of claim 68, wherein the exogenous exon comprises a CAP site. 70. The cell of claim 69, wherein the exogenous exon further comprises the nucleotide sequence ATG. 71. The cell of claim 68, wherein the exogenous exon comprises a coding sequence in-frame with what prior to homologous recombination was the second exon of the endogenous gene. 72. The cell of claim 71, wherein the coding sequence of the exogenous exon encodes the same amino acid sequence as that encoded by the first exon of erythropoietin. 73. The cell of claim 71, wherein the coding sequence of the exogenous exon is different from the coding sequence of the first exon of erythropoietin. 74. The cell of claim 71, wherein the coding sequence of the exogenous exon encodes the same amino acid sequence as that encoded by the first exon of human growth hormone. 75. The cell of claim 68, wherein what prior to homologous recombination was the first endogenous exon of the endogenous gene is deleted. 76. The cell of claim 68, wherein the endogenous gene encodes a hormone, a cytokine, an antigen, an antibody, an enzyme, a clotting factor, a transport protein, a receptor, a regulatory protein, a structural protein, or a transcription factor. 77. The cell of claim 68, wherein the endogenous gene encodes a protein selected from the group consisting of calcitonin, insulin, insulinotropin, insulin-like growth factors, parathyroid hormone, nerve growth factors, TGF-.beta., tumor necrosis factor, glucagon, bone growth factor-2, bone growth factor-7, TSH-.beta., interleukin 1, interleukin 2, interleukin 3, interleukin 6, interleukin 11, interleukin 12, CSF-macrophage, immunoglobulins, catalytic antibodies, protein kinase C, superoxide dismutase, tissue plasminogen activator, urokinase, antithrombin III, DNase, tyrosine hydroxylase, blood clotting factor V, blood clotting factor VII, blood clotting factor VIII, blood clotting factor X, blood clotting factor XIII, apolipoprotein E, apolipoprotein A-I, globins, low density lipoprotein receptor, IL-2 receptor, IL-2 receptor antagonists, alpha-1 antitrypsin, immune response modifiers, and soluble CD4. 78. The cell of claim 68, wherein the endogenous gene encodes growth hormone. 79. The cell of claim 68, wherein the endogenous gene encodes blood clotting factor IX. 80. The cell of claim 68, wherein the endogenous gene encodes .alpha.-galactosidase. 81. The cell of claim 68, wherein the endogenous gene encodes glucocerebrosidase. 82. The cell of claim 68, wherein the endogenous gene encodes erythropoietin. 83. The cell of claim 68, wherein the coding sequence of the exogenous exon is the same as the coding sequence of the first exon of the endogenous gene. 84. The cell of claim 68, wherein the coding sequence of the exogenous exon is different from the coding sequence of the first exon of the endogenous gene. 85. The cell of claim 68, wherein the exogenous regulatory sequence, exogenous exon and splice-donor site are upstream of the coding sequence of the endogenous gene. 86. The cell of claim 68, wherein the cell expresses a therapeutic protein. 87. The cell of claim 86, wherein the cell expresses erythropoietin. 88. The cell of claim 68, wherein the cell expresses a fusion protein comprising a first amino acid sequence encoded by the exogenous exon and a second amino acid sequence encoded by a portion of the endogenous gene. 89. The cell of claim 88, wherein the endogenous gene encodes erythropoietin. 90. The cell of claim 89, wherein the fusion protein comprises amino acids 1-3 of human growth hormone signal peptide. 91. The cell of claim 89, wherein the erythropoietin is human erythropoietin. 92. The cell of claim 68, wherein the cell is a primary or secondary cell of vertebrate origin. 93. The cell of claim 92, wherein the cell is a primary or secondary mammalian cell. 94. The cell of claim 92, wherein the cell is a primary or secondary human cell. 95. The cell of claim 68, wherein the cell is an immortalized mammalian cell. 96. The cell of claim 68, wherein the cell is an immortalized human cell. 97. The cell of claim 68, wherein the cell is selected from the group consisting of: HeLa cells and derivatives of HeLa cells, MCF-7 breast cancer cells, K-562 leukemia cells, KB carcinoma cells, 2780AD ovarian carcinoma cells, Raji cells, Jurkat cells, Namalwa cells, HL-60 cells, Daudi cells, RPMI 2426 cells, U-937 cells, Bowes Melanoma cells, WI-38VA13 subline 2R4 cells, and MOLT-4 cells. 98. The cell of claim 68, wherein the cell is an HT1080 cell. 99. The cell of claim 68, wherein the exogenous regulatory sequence is a promoter, an enhancer, a scaffold-attachment region or a transcription factor binding site. 100. The cell of claim 99, wherein the exogenous regulatory sequence is a promoter. 101. The cell of claim 99, wherein the exogenous regulatory sequence is a regulatory sequence of an adenovirus gene, a regulatory sequence of an SV-40 gene, or a regulatory sequence of a cytomegalovirus gene. 102. The cell of claim 99, wherein the exogenous regulatory sequence is a regulatory sequence of a mouse metallothionein gene, a regulatory sequence of a collagen gene, a regulatory sequence of an actin gene, a regulatory sequence of an immunoglobulin gene, a regulatory sequence of an HMG-CoA reductase gene, or a regulatory sequence of an EF-1.alpha. gene. 103. A cultured vertebrate cell the genome of which bears a transcription unit comprising an exogenous regulatory sequence, an exogenous exon and a splice-donor site at the 3' end of the exogenous exon, all located upstream of the transcription initiation site of an endogenous gene in a chromosome of the cell, the splice-donor site being operatively linked to the endogenous splice-acceptor site of the second endogenous exon of the endogenous gene. 104. A DNA construct which alters the expression of an targeted gene in a cell when the DNA construct is homologously recombined with a target site within the chromosomal DNA of the cell, the DNA construct comprising: (a) a targeting sequence homologous to the target site; (b) an exogenous regulatory sequence; (c) an exon; (d) a splice-donor site; (e) an intron; and (f) a splice-acceptor site; wherein upon homologous recombination, (b)-(f) are present in the chromosomal DNA of the cell in addition to all exons of the targeted gene, and the exogenous regulatory sequence controls transcription of (c)-(f) as well as part or all of the targeted gene. 105. The DNA construct of claim 104, wherein the target site is upstream of an endogenous regulatory sequence of the targeted gene. 106. The DNA construct of claim 104, wherein the construct further comprises a second targeting sequence homologous to a sequence upstream of an endogenous regulatory sequence of the targeted gene. 107. The DNA construct of claim 104, wherein the targeted gene encodes a therapeutic protein. 108. The DNA construct of claim 104, wherein the targeted gene encodes a hormone, a cytokine, an antigen, an antibody, an enzyme, a clotting factor, a transport protein, a receptor, a regulatory protein, a structural protein, or a transcription factor. 109. The DNA construct of claim 104, wherein the targeted gene encodes a protein selected from the group consisting of calcitonin, insulin, insulinotropin, insulin-like growth factors, parathyroid hormone, nerve growth factors, TGF-.beta., tumor necrosis factor, glucagon, bone growth factor-2, bone growth factor-7, TSH-.beta., interleukin 1, interleukin 2, interleukin 3, interleukin 6, interleukin 11, interleukin 12, CSF-macrophage, immunoglobulins, catalytic antibodies, protein kinase C, glucocerebrosidase, superoxide dismutase, tissue plasminogen activator, urokinase, antithrombin III, DNase, tyrosine hydroxylase, blood clotting factor V, blood clotting factor VII, blood clotting factor VIII, blood clotting factor IX, blood clotting factor X, blood clotting factor XIII, apolipoprotein E, apolipoprotein A-I, globins, low density lipoprotein receptor, IL-2 receptor, IL-2 receptor antagonists, alpha-1 antitrypsin, immune response modifiers, soluble CD4, erythropoietin, and growth hormone. 110. The DNA construct of claim 104, wherein the targeted gene encodes .alpha.-galactosidase. 111. The DNA construct of claim 104, wherein the targeted gene encodes .alpha.-interferon. 112. The DNA construct of claim 104, wherein the exogenous regulatory sequence is a promoter, an enhancer, a scaffold-attachment region, or a transcription factor binding site. 113. The DNA construct of claim 112, further comprising a second regulatory sequence. 114. The DNA construct of claim 112, wherein the exogenous regulatory sequence is a regulatory sequence of an adenovirus gene, a regulatory sequence of an SV-40 gene, or a regulatory sequence of a cytomegalovirus gene. 115. The DNA construct of claim 112, wherein the regulatory sequence is a regulatory sequence of a mouse metallothionein-I gene, a regulatory sequence of a collagen gene, a regulatory sequence of an actin gene, a regulatory sequence of an immunoglobulin gene, a regulatory sequence of an HMG-CoA reductase gene, or a regulatory sequence of an EF-1.alpha. gene. 116. The DNA construct of claim 104, further comprising a gene. 117. The DNA construct of claim 104, further comprising one or more selectable marker genes. 118. The DNA construct of claim 117, further comprising an amplifiable marker gene. 119. A method of altering the expression of a targeted gene in a cell, comprising the steps of: (a) transfecting the cell with a DNA construct, the DNA construct comprising: (i) a targeting sequence; (ii) an exogenous regulatory sequence; (iii) an exon; (iv) a splice-donor site; (v) an intron; and (vi) a splice-acceptor site, to generate a transfected cell; (b) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell the chromosomal DNA of which comprises the construct-derived exon and intron in addition to the endogenous coding sequence of the targeted gene; and (c) maintaining the homologously recombinant cell under conditions appropriate for transcription of (iii)-(vi) and the targeted gene under the control of the exogenous regulatory sequence, thereby producing a transcript. 120. The method of claim 119, wherein the size of the intron is selected to maximize expression of the targeted gene under the control of the exogenous regulatory sequence. 121. The method of claim 119, wherein the exon comprises a CAP site. 122. The method of claim 121, wherein the exon comprises the nucleotide sequence ATG. 123. A fusion protein produced by the method of claim 122 and containing a first amino acid sequence encoded by the construct-derived exon and a second amino acid sequence encoded by the entire targeted gene. 124. The method of claim 119, wherein the targeting sequence is homologous to a sequence upstream of an endogenous regulatory sequence of the targeted gene. 125. The method of claim 119, wherein the construct further comprises a second targeting sequence homologous to a sequence upstream of an endogenous regulatory sequence of the targeted gene. 126. The method of claim 119, wherein the cell is a human cell. 127. The method of claim 119, wherein the targeted gene encodes a therapeutic protein. 128. The method of claim 119, wherein the targeted gene encodes a hormone, a cytokine, an antigen, an antibody, an enzyme, a clotting factor, a transport protein, a receptor, a regulatory protein, a structural protein, or a transcription factor. 129. The method of claim 119, wherein the targeted gene encodes a protein selected from the group consisting of calcitonin, insulin, insulinotropin, insulin-like growth factors, parathyroid hormone, .beta.-interferon, .gamma.-interferon, nerve growth factors, TGF-.beta., tumor necrosis factor, glucagon, bone growth factor-2, bone growth factor-7, TSH-.beta., interleukin 1, interleukin 2, interleukin 3, interleukin 6, interleukin 11, interleukin 12, CSF-macrophage, immunoglobulins, catalytic antibodies, protein kinase C, glucocerebrosidase, superoxide dismutase, tissue plasminogen activator, urokinase, antithrombin III, DNase, tyrosine hydroxylase, blood clotting factor V, blood clotting factor VII, blood clotting factor VIII, blood clotting factor IX, blood clotting factor X, blood clotting factor XIII, apolipoprotein E, apolipoprotein A-I, a globin, low density lipoprotein receptor, IL-2 receptor, IL-2 receptor antagonists, alpha-1 antitrypsin, immune response modifiers, soluble CD4, erythropoietin, and growth hormone. 130. The method of claim 119, wherein the targeted gene encodes .alpha.-galactosidase. 131. The method of claim 119, wherein the targeted gene encodes .alpha.-interferon. 132. The method of claim 119, wherein the exogenous regulatory sequence is a promoter, an enhancer, a scaffold-attachment region, or a transcription factor binding site. 133. The method of claim 132, wherein the construct further comprises a second regulatory sequence. 134. The method of claim 132, wherein the exogenous regulatory sequence is a regulatory sequence of an adenovirus gene, a regulatory sequence of an SV-40 gene, or a regulatory sequence of a cytomegalovirus gene. 135. The method of claim 132, wherein the exogenous regulatory sequence is a regulatory sequence of a mouse metallothionein-I gene, a regulatory sequence of a collagen gene, a regulatory sequence of an actin gene, a regulatory sequence of an immunoglobulin gene, a regulatory sequence of an HMG-CoA reductase gene, or a regulatory sequence of an EF-1.alpha. gene. 136. The method of claim 119, further comprising the steps of: (f) maintaining the homologously recombinant cell under conditions appropriate for splicing and translation of the transcript; and (g) confirming that a translation product of the transcript was produced. 137. The method of claim 136 in which the targeted gene encodes .alpha.-interferon. 138. The method of claim 136 in which the targeted gene encodes .alpha.-galactosidase. 139. A cultured vertebrate cell having incorporated therein a transcription unit comprising an exogenous regulatory sequence, an exogenous exon, a splice-donor site, an intron, and a splice-acceptor site, wherein the exogenous regulatory sequence is positioned to control transcription of the exogenous exon, splice-donor site, intron, and splice-acceptor site, in addition to all endogenous exons of an endogenous gene. 140. The cell of claim 139, wherein the exogenous exon comprises a CAP site. 141. The cell of claim 139, wherein the exogenous regulatory sequence, exogenous exon and splice-donor site are upstream of the coding sequence of the endogenous gene. 142. The cell of claim 139, wherein an endogenous regulatory sequence of the endogenous gene is deleted. 143. The cell of claim 139, wherein the first endogenous exon of the endogenous gene is deleted. 144. The cell of claim 139, wherein the endogenous gene encodes a hormone, a cytokine, an antigen, an antibody, an enzyme, a clotting factor, a transport protein, a receptor, a regulatory protein, a structural protein, or a transcription factor. 145. The cell of claim 139, wherein the endogenous gene encodes a protein selected from the group consisting of erythropoietin, calcitonin, growth hormone, insulin, insulinotropin, an insulin-like growth factor, parathyroid hormone, .beta.-interferon, .gamma.-interferon, nerve growth factors, FSH.beta., TGF-.beta., tumor necrosis factor, glucagon, bone growth factor-2, bone growth factor-7, TSH-.beta., interleukin 1, interleukin 2, interleukin 3, interleukin 6, interleukin 11, interleukin 12, CSF-granulocyte, CSF-macrophage, CSF-granulocyte/macrophage, immunoglobulins, catalytic antibodies, protein kinase C, glucocerebrosidase, superoxide dismutase, tissue plasminogen activator, urokinase, antithrombin III, DNase, tyrosine hydroxylase, blood clotting factor V, blood clotting factor VII, blood clotting factor VIII, blood clotting factor IX, blood clotting factor X, blood clotting factor XIII, apolipoprotein E, apolipoprotein A-I, globins, low density lipoprotein receptor, IL-2 receptor, IL-2 receptor antagonists, alpha-1 antitrypsin, immune response modifiers, soluble CD4, growth hormone, and erythropoietin. 146. The cell of claim 139, wherein the endogenous gene encodes .alpha.-interferon. 147. The cell of claim 139, wherein the endogenous gene encodes .alpha.-galactosidase. 148. The cell of claim 139, wherein the cell is a primary or secondary cell of vertebrate origin. 149. The cell of claim 148, wherein the cell is a primary or secondary mammalian cell. 150. The cell of claim 148, wherein the cell is a primary or secondary human cell. 151. The cell of claim 148, wherein the cell is an immortalized mammalian cell. 152. The cell of claim 148, wherein the cell is an immortalized human cell. 153. The cell of claim 139, wherein the cell is selected from the group consisting of: HeLa cells and derivatives of HeLa cells, MCF-7 breast cancer cells, K-562 leukemia cells, KB carcinoma cells, 2780AD ovarian carcinoma cells, Raji cells, Jurkat cells, Namalwa cells, HL-60 cells, Daudi cells, RPMI 2426 cells, U-937 cells, Bowes Melanoma cells, WI-38VA13 subline 2R4 cells, and MOLT-4 cells. 154. The cell of claim 139, wherein the cell is an HT1080 cell. 155. The cell of claim 139, wherein the cell expresses a therapeutic protein. 156. The cell of claim 139, wherein the cell expresses .alpha.-interferon. 157. The cell of claim 139, wherein the cell expresses .alpha.-galactosidase. 158. The cell of claim 139, wherein the exogenous regulatory sequence is a promoter, an enhancer, a scaffold-attachment region, or a transcription factor binding site. 159. The cell of claim 158, wherein the exogenous regulatory sequence is a promoter. 160. The cell of claim 158, wherein the exogenous regulatory sequence is a regulatory sequence of an adenovirus gene, a regulatory sequence of an SV-40 gene, or a regulatory sequence of a cytomegalovirus gene. 161. The cell of claim 158, wherein the exogenous regulatory sequence is a regulatory sequence of a mouse metallothionein gene, a regulatory sequence of a collagen gene, a regulatory sequence of an actin gene, a regulatory sequence of an immunoglobulin gene, a regulatory sequence of an HMG-CoA reductase gene, or a regulatory sequence of an EF-1.alpha. gene. 162. The method of making a homologously recombinant cell wherein the expression of a targeted gene is altered, comprising the steps of: (a) transfecting a cell with a DNA construct, the construct comprising: (i) a targeting sequence homologous to a target site in the chromosomal DNA of the cell; (ii) an exogenous regulatory sequence; (iii) an exon; (iv) a splice-donor site; (v) an intron; and (vi) a splice-acceptor site, to generate a transfected cell; and (b) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell the chromosomal DNA of which comprises the construct-derived exon and intron in addition to the endogenous sequence of the targeted gene, wherein the exogenous regulatory sequence controls expression of a transcript comprising RNA corresponding to (iii)-(vi) and part or all of the targeted gene. 163. A homologously recombinant cell produced by the method of claim 162. 164. A method of altering the expression of a targeted gene in a cell, comprising the steps of: (a) transfecting a cell with a DNA construct, the construct comprising: (i) a targeting sequence; (ii) an exogenous regulatory sequence; (iii) an exon; (iv) a splice-donor site; (v) an intron; and (vi) a splice-acceptor site, thereby producing a transfected cell, wherein the targeting sequence directs the integration of elements (ii)-(vi) into genomic DNA of the cell such that the regulatory sequence is positioned to direct transcription of a sequence comprising (iii)-(vi) and sequence of the targeted gene; (b) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell; and (c) maintaining the homologously recombinant cell under conditions appropriate for expression under the control of the regulatory sequence. 165. A method for expressing an erythropoietin gene in the genome of a mammalian cell, comprising transfecting the cell with a construct comprising (a) a targeting sequence, (b) an exogenous regulatory sequence, (c) an exon; and (d) an unpaired splice-donor site at the 3' end of the exon, wherein the targeting sequence directs the integration of (b)-(d) into the chromosomal DNA of the cell upstream and within 30 kb of the endogenous ATG initiation codon of the erythropoietin gene such that the exogenous regulatory sequence directs expression of a transcript comprising sequence corresponding to the construct-derived exon and all endogenous exons of the erythropoietin gene. 166. The method of claim 165, wherein the targeting sequence is homologous to a sequence located between about 5 kilobases and about 30 kilobases upstream of the ATG initiation codon of the erythropoietin gene. 167. The method of claim 165, wherein the mammalian cell is a human cell. 168. A cultured vertebrate cell which expresses erythropoietin, the genome of said cell having incorporated therein a transcription unit comprising an exogenous regulatory region, an exogenous exon, and an exogenous splice-donor site, wherein transcription under the control of the exogenous regulatory region produces a transcript comprising RNA corresponding to the exogenous exon, the exogenous splice-donor site, and all endogenous exons of an endogenous erythropoietin gene, wherein the RNA corresponding to the splice-donor site directs splicing to a splice-acceptor site of the transcript which corresponds to a site within the endogenous erythropoietin gene. 169. The cultured vertebrate cell of claim 168, wherein the splice-acceptor site of the transcript corresponds to the splice-acceptor site of the second exon of the erythropoietin gene. 170. A cultured vertebrate cell comprising the dhfr gene, the neo gene, the CMV immediate early promoter, intron 1 of the CMV immediate early gene, hGH exon 1 and an unpaired splice-donor site inserted at a position upstream of the ATG initiation codon of an endogenous erythropoietin gene, such that after transcription driven by the CMV promoter, the hGH exon 1 portion of the transcript is spliced to the portion of the transcript transcribed from exon 2 of the erythropoietin gene. 171. The cultured vertebrate cell of claim 170 produced by the integration of DNA from pREPO18. 172. A DNA construct which alters expression of an endogenous GM-CSF (colony stimulating factor-granulocyte/macrophage) gene in a cell when the DNA construct is homologously recombined with a target site within or upstream of a GM-CSF gene in the chromosomal DNA of the cell, the construct comprising: (a) a targeting sequence homologous with the target site; (b) an exogenous regulatory sequence; (c) an exon; and (d) an unpaired splice-donor site at the 3' end of the exon; wherein following homologous recombination of the targeting sequence with the target site, the exogenous regulatory sequence controls expression of a transcript comprising RNA corresponding to the construct-derived exon in addition to all endogenous exons of the GM-CSF gene. 173. The DNA construct of claim 172, wherein (a)-(d) are oriented such that, upon integration of the construct into chromosomal DNA of the cell at the target site, (b)-(d) are integrated upstream of exon 1 of the endogenous GM-CSF gene, and a transcript produced under the control of the exogenous regulatory sequence of (b) contains a splice-donor site (i) that corresponds to the splice-donor site of (d), and (ii) is spliced to an endogenous splice-acceptor site of the second endogenous exon of the GM-CSF gene. 174. The DNA construct of claim 173, wherein the construct-derived exon of the transcript comprises coding sequence which, upon splicing of the construct-derived splice-donor site of the transcript with the endogenous splice-acceptor site of the transcript, is in-frame with the RNA corresponding to the coding sequence of the GM-CSF gene. 175. The DNA construct of claim 174, wherein the coding sequence of the construct-derived exon encodes the same amino acid sequence as encoded by the first endogenous exon of the GM-CSF gene. 176. The DNA construct of claim 174, wherein the coding sequence of the construct-derived exon is different from the coding sequence of the first endogenous exon of the GM-CSF gene. 177. The DNA construct of claim 172, wherein the exogenous regulatory sequence comprises a promoter. 178. The DNA construct of claim 172, further comprising a DNA sequence encoding a selectable marker. 179. The DNA construct of claim 172, further comprising an amplifiable marker gene. 180. The DNA construct of claim 172, wherein the construct-derived exon comprises a CAP site. 181. The DNA construct of claim 180, wherein the construct-derived exon further comprises the nucleotide sequence ATG. 182. The DNA construct of claim 172, wherein the targeting sequence is homologous to a sequence within the GM-CSF gene. 183. The DNA construct of claim 172, wherein the targeting sequence is homologous to a sequence upstream of the coding region of the GM-CSF gene. 184. The DNA construct of claim 172, wherein the targeting sequence is homologous to a sequence upstream of an endogenous regulatory region of the GM-CSF gene. 185. The DNA construct of claim 172, further comprising a second targeting sequence homologous to a second target site within or upstream of the endogenous GM-CSF coding sequence. 186. The DNA construct of claim 172, wherein the construct-derived exon encodes the first 50 amino acids of the GM-CSF precursor protein. 187. The DNA construct of claim 172, wherein the construct-derived exon encodes a sequence comprising a functional signal peptide. 188. A method of producing a homologously recombinant cell wherein the expression of a GM-CSF gene is altered, comprising the steps of: (a) providing a cell, the genome of which contains an endogenous GM-CSF gene; (b) transfecting the cell with a DNA construct, the construct comprising (i) a targeting sequence homologous with a target site within or upstream of the endogenous GM-CSF gene; (ii) an exogenous regulatory sequence; (iii) an exon; and (iv) an unpaired splice-donor site at the 3' end of the exon, thereby producing a transfected cell; and (c) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell in which the exogenous regulatory sequence controls expression of a transcript comprising RNA corresponding to the construct-derived exon, the construct-derived splice-donor site, and all endogenous exons of the GM-CSF gene. 189. The method of claim 188, wherein the cell is of vertebrate origin. 190. The method of claim 189, wherein the cell is a primary or secondary mammalian cell. 191. The method of claim 189, wherein the cell is a primary or secondary human cell. 192. The method of claim 189, wherein the cell is an immortalized mammalian cell. 193. The method of claim 189, wherein the cell is an immortalized human cell. 194. The method of claim 188, wherein the cell is selected from the group consisting of: HeLa cells and derivatives of HeLa cells, MCF-7 breast cancer cells, K-562 leukemia cells, KB carcinoma cells, 2780AD ovarian carcinoma cells, Raji cells, Jurkat cells, Namalwa cells, HL-60 cells, Daudi cells, RPMI 2426 cells, U-937 cells, Bowes Melanoma cells, WI-38VA13 subline 2R4 cells, and MOLT-4 cells. 195. The method of claim 188, wherein the cell is an HT1080 cell. 196. A homologously recombinant cell produced by the method of claim 188. 197. The cell of claim 68, wherein the exogenous regulatory sequence, exogenous exon and splice-donor site are upstream of an endogenous regulatory sequence of the endogenous gene. 198. The cell of claim 68, wherein what prior to homologous recombination was an endogenous regulatory sequence of the endogenous gene is deleted. 199. A method for producing GM-CSF, comprising the steps of: (a) transfecting a cell, the chromosomal DNA of which contains an endogenous GM-CSF gene, with a DNA construct comprising (i) a targeting sequence homologous with a target site within or upstream of the endogenous GM-CSF gene; (ii) an exogenous regulatory sequence; (iii) an exon; and (iv) an unpaired splice-donor site at the 3' end of the exon, thereby producing a transfected cell; (b) maintaining the transfected cell under conditions appropriate for homologous recombination to occur, thereby producing a homologously recombinant cell in the genomic DNA of which (a)(ii)-(iv) are positioned upstream of the endogenous transcription initiation site of the GM-CSF gene, and the exogenous regulatory sequence controls expression of a transcript comprising RNA corresponding to (a)(iii)-(iv) and the GM-CSF gene; and (c) maintaining the homologously recombinant cell produced in step (b) under conditions appropriate for the production of GM-CSF under the control of the exogenous regulatory sequence. 200. A DNA construct which alters the expression of an endogenous G-CSF (colony stimulating factor-granulocyte) gene in a cell when the DNA construct is homologously recombined with a target site within or upstream of the coding region of the G-CSF gene in the chromosomal DNA of the cell, said construct comprising: (a) a targeting sequence homologous with the target site; (b) an exogenous regulatory sequence; (c) an exon; and (d) an unpaired splice-donor site at the 3' end of the exon; wherein following homologous recombination of the targeting sequence with the target site, the exogenous regulatory sequence controls expression of a transcript comprising RNA corresponding to the construct-derived exon in addition to all endogenous exons of the G-CSF gene. 201. The DNA construct of claim 200, wherein (a)-(d) are oriented such that, upon integration of the construct into chromosomal DNA of the cell at the target site, (b)-(d) are integrated upstream of exon 1 of the endogenous G-CSF gene and the transcript contains a splice-donor site that (i) corresponds to the splice-donor site of (d), and (ii) is spliced to an endogenous splice-acceptor site of the second endogenous exon of the G-CSF gene. 202. The DNA construct of claim 201, wherein the construct-derived exon of the transcript comprises coding sequence which, upon splicing of the construct-derived splice-donor site of the transcript with the endogenous splice-acceptor site of the transcript, is in-frame with RNA corresponding to the coding sequence of the G-CSF gene. 203. The DNA construct of claim 202, wherein the coding sequence of the construct-derived exon encodes the same amino acid sequence as that encoded by the coding sequence of the first endogenous exon of the G-CSF gene. 204. The DNA construct of claim 202, wherein the coding sequence of the construct-derived exon is different from the coding sequence of the first endogenous exon of the G-CSF gene. 205. The DNA construct of claim 200, wherein the exogenous regulatory sequence comprises a promoter. 206. The DNA construct of claim 200, further comprising a selectable marker gene. 207. The DNA construct of claim 206, further comprising an amplifiable marker gene. 208. The DNA construct of claim 200, wherein the construct-derived exon comprises a CAP site. 209. The DNA construct of claim 208, wherein the construct-derived exon further comprises the nucleotide sequence ATG. 210. The DNA construct of claim 200, wherein the target site is within the coding region of the G-CSF gene. 211. The DNA construct of claim 200 wherein the target site is upstream of the coding region of the G-CSF gene. 212. The DNA construct of claim 200, wherein the target site is upstream of an endogenous regulatory region of the G-CSF gene. 213. The DNA construct of claim 200 further comprising a second targeting sequence homologous to a second target site within or upstream of the endogenous G-CSF coding sequence. 214. The DNA construct of claim 200 wherein the construct-derived exon encodes an amino acid sequence which is identical to the first 13 amino acid residues of the G-CSF signal peptide. 215. The DNA construct of claim 200, wherein the construct-derived exon encodes an amino acid sequence which is functionally equivalent to the first 13 amino acids of the G-CSF signal peptide. 216. A method of producing a homologously recombinant cell wherein the expression of a G-CSF gene is altered, comprising the steps of: (a) providing a cell, the genome of which contains an endogenous G-CSF gene; (b) transfecting the cell with a DNA construct comprising (i) a targeting sequence homologous with a target site within or upstream of the endogenous G-CSF gene; (ii) an exogenous regulatory sequence; (iii) an exon; and (iv) an unpaired splice-donor site at the 3' end of the exon, thereby producing a transfected cell; and (c) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell in which the exogenous regulatory sequence controls expression of a transcript comprising RNA corresponding to the construct-derived exon, construct-derived splice-donor site, and all endogenous exons of the G-CSF gene. 217. The method of claim 216, wherein the cell is of vertebrate origin. 218. The method of claim 217, wherein the cell is a primary or secondary mammalian cell. 219. The method of claim 217, wherein the cell is a primary or secondary human cell. 220. The method of claim 217, wherein the cell is an immortalized mammalian cell. 221. The method of claim 217, wherein the cell is an immortalized human cell. 222. The method of claim 216, wherein the cell is selected from the group consisting of: HeLa cells and derivatives of HeLa cells, MCF-7 breast cancer cells, K-562 leukemia cells, KB carcinoma cells, 2780AD ovarian carcinoma cells, Raji cells, Jurkat cells, Namalwa cells, HL-60 cells, Daudi cells, RPMI 2426 cells, U-937 cells, Bowes Melanoma cells, WI-38VA13 subline 2R4 cells, and MOLT-4 cells. 223. The method of claim 216, wherein the cell is an HT1080 cell. 224. A homologously recombinant cell produced by the method of claim 216. 225. A cultured vertebrate cell which expresses G-CSF, the genome of the cell having incorporated therein a transcription unit comprising an exogenous regulatory region, an exogenous exon, and an exogenous splice-donor site at the 3' end of the exogenous exon, wherein transcription under the control of the exogenous regulatory region produces a transcript comprising RNA corresponding to the exogenous exon, the exogenous splice-donor site, and all of the endogenous exons of an endogenous G-CSF gene, wherein the RNA corresponding to the splice-donor site directs splicing to a splice-acceptor site of the transcript which corresponds to a site within the endogenous G-CSF gene. 226. The cell of claim 225, wherein the exogenous splice-donor site is operatively linked to the endogenous splice-acceptor site of the second exon of the G-CSF gene. 227. A method for producing G-CSF, comprising the steps of: (a) transfecting a cell, the chromosomal DNA of which contains an endogenous G-CSF gene, with a DNA construct comprising, (i) a targeting sequence homologous with a target site within or upstream of the endogenous G-CSF gene; (ii) an exogenous regulatory sequence; (iii) an exon; and (iv) an unpaired splice-donor site at the 3' end of the exon, thereby producing a transfected cell; (b) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell in the genomic DNA of which (a)(ii)-(iv) are positioned upstream of the endogenous transcription initiation site of the G-CSF gene, and the exogenous regulatory sequence controls expression of a transcript comprising RNA corresponding to (a)(iii)-(iv) and the G-CSF gene; and (c) maintaining the homologously recombinant cell produced in step (b) under conditions appropriate for the production of G-CSF under the control of the exogenous regulatory sequence. 228. A DNA construct which alters the expression of an FSH.beta. (follicle stimulating hormone-.beta.) gene in a cell when the DNA construct is homologously recombined with a target site within or upstream of an FSH.beta. gene in the chromosomal DNA of the cell, the construct comprising: (a) a targeting sequence homologous with the target site; (b) an exogenous regulatory sequence; (c) an exon; and (d) an unpaired splice-donor site at the 3' end of the exon, wherein following homologous recombination of the targeting sequence with the target site, the exogenous regulatory sequence controls expression of a transcript comprising RNA corresponding to the construct-derived exon in addition to all endogenous exons of the FSH.beta. gene. 229. The DNA construct of claim 228, wherein (a)-(d) are oriented such that, upon integration of the construct into chromosomal DNA of the cell at the target site, (b)-(d) are integrated within or upstream of intron 1 of the endogenous FSH.beta. gene, and a transcript produced under the control of the exogenous regulatory sequence of (b) contains a splice-donor site (i) that corresponds to the splice-donor site of (d), and (ii) is spliced to an endogenous splice-acceptor site of the second endogenous exon of the FSH.beta. gene. 230. The DNA construct of claim 229, wherein the exogenous regulatory sequence comprises a promoter. 231. The DNA construct of claim 229, further comprises a selectable marker gene. 232. The DNA construct of claim 229, further comprising an amplifiable marker gene. 233. The DNA construct of claim 229, wherein the exon comprises a CAP site. 234. The DNA construct of claim 229, wherein the construct-derived exon is the same as the first exon of the FSH.beta. gene. 235. The DNA construct of claim 229, wherein the construct-derived exon is different from the first exon of the FSH.beta. gene. 236. The DNA construct of claim 229, wherein the target site is upstream of an endogenous regulatory region of the FSH.beta. gene. 237. The DNA construct of claim 229, further comprising a second targeting sequence homologous to a second target site within or upstream of the endogenous FSH.beta. gene. 238. A DNA construct which alters the expression of an FSH.beta. (follicle stimulating hormone-.beta.) gene in a cell when the DNA construct is homologously recombined with a target site within or upstream of the first intron of an FSH.beta. gene in the chromosomal DNA of the cell, the construct comprising: (a) a targeting sequence homologous with the target site; (b) an exogenous regulatory sequence; (c) an exon; and (d) an unpaired splice-donor site at the 3' end of the exon, wherein following homologous recombination of the targeting sequence with the target site, the exogenous regulatory sequence controls expression of a transcript comprising RNA corresponding to the construct-derived exon in addition to all endogenous exons of the FSH.beta. gene. 239. A method of producing a homologously recombinant cell wherein the expression of an FSH.beta. gene is altered, comprising the steps of: (a) providing a cell, the genome of which contains an endogenous FSH.beta. gene; (b) transfecting the cell with a DNA construct comprising, (i) a targeting sequence homologous with a target site within or upstream of the endogenous FSH.beta. gene; (ii) an exogenous regulatory sequence; (iii) an exon; and (iv) an unpaired splice-donor site at the 3' end of the exon, thereby producing a transfected cell; and (c) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell in which the exogenous regulatory sequence controls expression of a transcript comprising RNA corresponding to the construct-derived exon, the construct-derived splice-donor site, and all of the endogenous exons of the FSH.beta. gene. 240. The method of claim 239, wherein the cell is of vertebrate origin. 241. The method of claim 240, wherein the cell is a primary or secondary mammalian cell. 242. The method of claim 240, wherein the cell is a primary or secondary human cell. 243. The method of claim 240, wherein the cell is an immortalized mammalian cell. 244. The method of claim 240, wherein the cell is an immortalized human cell. 245. The method of claim 244, wherein the cell is selected from the group consisting of: HeLa cells and derivatives of HeLa cells, MCF-7 breast cancer cells, K-562 leukemia cells, KB carcinoma cells, 2780AD ovarian carcinoma cells, Raji cells, Jurkat cells, Namalwa cells, HL-60 cells, Daudi cells, RPMI 2426 cells, U-937 cells, Bowes Melanoma cells, WI-38VA13 subline 2R4 cells, and MOLT-4 cells. 246. The method of claim 244, wherein the cell is an HT1080 cell. 247. A homologously recombinant cell produced by the method of claim 239. 248. An isolated DNA molecule comprising a sequence selected from the group consisting of SEQ ID NO:3 and a fragment of SEQ ID NO:3 which selectively promotes homologous recombination with genomic DNA upstream of a thrombopoietin gene. 249. A method for producing FSH.beta., comprising the steps of: (a) transfecting a cell, the chromosomal DNA of which contains an endogenous FSH.beta. gene, with a DNA construct comprising (i) a targeting sequence homologous with a target site within or upstream of the endogenous FSH.beta. gene; (ii) an exogenous regulatory sequence; (iii) an exon; and (iv) an unpaired splice-donor site at the 3' end of the exon, thereby producing a transfected cell; (b) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell in the genomic DNA of which (a)(ii)-(iv) are positioned upstream of the endogenous transcription initiation site of the FSH.beta. gene, and the exogenous regulatory sequence controls expression of a transcript comprising RNA corresponding to (a)(iii)-(iv) and the FSH.beta. gene; and (c) maintaining the homologously recombinant cell produced in step (b) under conditions appropriate for the production of FSH.beta. under the control of the exogenous regulatory sequence. 250. The method of claim 249, wherein the cell as obtained expresses human glycoprotein .alpha.-subunit. 251. The method of claim 249, wherein the cell expresses an human glycoprotein .alpha.-subunit gene under the control of an exogenous promoter. 252. A DNA construct which alters the expression of a gene encoding thrombopoietin when inserted by homologous recombination into chromosomal DNA of a cell, said construct comprising: (a) a targeting sequence homologous with a target site upstream of the endogenous thrombopoietin gene; (b) a regulatory sequence; (c) a non-coding exon; and (d) an unpaired splice-donor site at the 3' end of the exon, wherein, following homologous recombination of the targeting sequence with the target site, exogenous regulatory sequence controls expression of a transcript comprising RNA corresponding to the construct-derived exon in addition to all endogenous exons of the thrombopoietin gene. 253. The DNA construct of claim 252, wherein the regulatory sequence comprises a promoter. 254. The DNA construct of claim 252, further comprising a selectable marker gene. 255. The DNA construct of claim 252, further comprising an amplifiable marker gene. 256. The DNA construct of claim 252, further comprising a second targeting sequence comprising DNA which selectively promotes homologous recombination with genomic DNA upstream of the thrombopoietin gene. 257. The DNA construct of claim 252 wherein the targeting sequence is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, fragments of SEQ ID NO:3 which selectively promote homologous recombination with genomic DNA upstream of the thrombopoietin gene and fragments of SEQ ID NO:4 which selectively promote homologous recombination with genomic DNA upstream of the thrombopoietin gene. 258. The DNA construct of claim 257 wherein the targeting sequence is fragment of SEQ ID NO:3 and is at least about 20 nucleotides. 259. The DNA construct of claim 257 wherein the targeting sequence is a fragment of SEQ ID NO:4 and is at least about 20 nucleotides. 260. The DNA construct of claim 257 wherein the targeting sequence is at least about 20 nucleotides and is a sequence between about nucleotides -1815 to -145, 14 to 245, or 374 to 570 of FIG. 5 (SEQ ID NO:4). 261. A method for producing thrombopoietin, comprising the steps of: (a) transfecting a cell containing the thrombopoietin gene with the DNA construct of claim 252; (b) maintaining the transfected cell under conditions appropriate for homologous recombination to occur; and (c) maintaining the homologously recombinant cell produced in step (b) under conditions appropriate for the expression of thrombopoietin under the control of the exogenous regulatory sequence. 262. An isolated DNA molecule comprising a sequence which is selected from the group consisting of about nucleotides -1815 to -145 of FIG. 5 (SEQ ID NO:4), about nucleotides 14 to 245 of FIG. 5 (SEQ ID NO:4), and about nucleotides 374 to 570 of FIG. 5 (SEQ ID NO:4), and which selectively promotes homologous recombination with genomic DNA within or upstream of a thrombopoietin gene. 263. A DNA construct which alters the expression of a gene encoding thrombopoietin when inserted by homologous recombination into chromosomal DNA of the cell, said construct comprising: (a) a targeting sequence homologous with a target site within or upstream of the endogenous thrombopoietin coding region; (b) a regulatory sequence; (c) a non-coding exon; and (d) an unpaired splice-donor site at the 3' end of the exon, wherein, following homologous recombination of the targeting sequence with the target site, the regulatory sequence of (b), the non-coding exon of (c), and the unpaired splice-donor site of (d) are integrated upstream of the first exon of the thrombopoietin gene and upon expression under the control of the regulatory sequence, a transcript is produced in which sequence corresponding to the construct-derived splice-donor site is spliced to sequence corresponding to the splice-acceptor site of the second endogenous exon of the thrombopoietin gene. 264. The DNA construct of claim 263, further comprising a selectable marker gene. 265. The DNA construct of claim 263, further comprising an amplifiable marker gene. 266. The DNA construct of claim 263, further comprising a second targeting sequence comprising DNA which selectively promotes homologous recombination with genomic DNA upstream of the thrombopoietin gene. 267. The DNA construct of claim 263, wherein the targeting sequence is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, fragments of SEQ ID NO:3 which selectively promote homologous recombination with genomic DNA upstream of the thrombopoietin gene and fragments of SEQ ID NO:4 which selectively promote homologous recombination with genomic DNA upstream of the thrombopoietin gene. 268. The DNA construct of claim 267, wherein the targeting sequence is a fragment of SEQ ID NO:4 and is at least about 30 nucleotides. 269. A method of producing a homologously recombinant cell wherein the expression of a thrombopoietin gene is altered, comprising the steps of: (a) transfecting a cell containing the thrombopoietin gene with a DNA construct comprising (i) a targeting sequence homologous with a target site within or upstream of the thrombopoietin gene; (ii) an exogenous regulatory sequence; (iii) a non-coding exon; and (iv) an unpaired splice-donor site at the 3' end of the exon, thereby producing a transfected cell, and (b) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell in which the exogenous regulatory sequence controls expression of a transcript comprising the construct-derived exon, the construct-derived splice-donor site, and all endogenous exons of the thrombopoietin gene. 270. A homologously recombinant cell produced by the method of claim 269. 271. A cultured vertebrate cell which expresses thrombopoietin, said cell having incorporated therein a transcription unit which comprises an exogenous regulatory sequence, an exogenous non-coding exon, and an exogenous splice-donor site, the splice-donor site being operatively linked to the endogenous splice-acceptor site of the second endogenous exon of he thrombopoietin gene, wherein the cell comprises the exogenous non-coding exon in addition to all endogenous exons of the thrombopoietin gene. 272. The cell of claim 271, wherein the exogenous splice-donor site is operatively linked to the endogenous splice-acceptor site of the second exon of the thrombopoietin gene. 273. A method for producing thrombopoietin comprising maintaining the cell of claim 271 under conditions appropriate for the expression of thrombopoietin under the control of the exogenous regulatory sequence. 274. A DNA construct which alters the expression of a gene encoding DNase I when inserted by homologous recombination into chromosomal DNA of the cell, said construct comprising: (a) a targeting sequence homologous with a target site within or upstream of the DNase I gene; (b) an exogenous regulatory sequence; (c) an exon; and (d) an unpaired splice-donor site at the 3' end of the exon, wherein following homologous recombination of the targeting sequence with the target site, the exogenous regulatory sequence controls expression of a transcript comprising RNA corresponding to the construct-derived exon in addition to all endogenous exons of the DNase I gene. 275. The DNA construct of claim 274, wherein the regulatory sequence comprises a promoter. 276. The DNA construct of claim 274, further comprising a selectable marker gene. 277. The DNA construct of claim 274, further comprising an amplifiable marker gene. 278. The DNA construct of claim 274, further comprising a second targeting sequence comprising DNA which hybridizes to genomic DNA within or upstream of the DNase I gene. 279. The DNA construct of claim 274 wherein the targeting sequence is selected from the group consisting of SEQ ID NO:17, SEQ ID NO: 18, fragments of SEQ ID NO:17 which selectively promote homologous recombination with genomic DNA upstream of the DNase I gene and fragments of SEQ ID NO:18 which selectively promote homologous recombination with genomic DNA within or upstream of the DNase I gene. 280. The DNA construct of claim 279 wherein the targeting sequence is a fragment of SEQ ID NO:17 and is at least about 20 nucleotides. 281. The DNA construct of claim 279 wherein the targeting sequence is a fragment of SEQ ID NO:18 and is at least about 20 nucleotides. 282. The DNA construct of claim 279 wherein the targeting sequence is at least about 20 base pairs and is a sequence between about nucleotides -328 to -2 of FIG. 11 (SEQ ID NO:18). 283. An isolated DNA molecule comprising a sequence selected from the group consisting of SEQ ID NO:17 and a fragment of SEQ ID NO:17 which selectively promotes homologous recombination with genomic DNA upstream of a DNase I gene. 284. An isolated DNA molecule of at least about 20 nucleotides which selectively promotes homologous recombination with genomic DNA within or upstream of the DNase I gene, the DNA molecule comprising a sequence selected from the group consisting of a sequence between about nucleotides -328 to -2 of FIG. 11 (SEQ ID NO:18) and a sequence which hybridizes to a sequence between about nucleotides -328 to -2 of FIG. 11 (SEQ ID NO:18). 285. A method of producing a homologously recombinant cell wherein the expression of a DNase I gene is altered, comprising the steps of: (a) transfecting a cell containing the DNase I gene with a DNA construct comprising (i) a targeting sequence homologous with a target site within or upstream of the DNase I gene; (ii) an exogenous regulatory sequence; (iii) an exon; and (iv) an unpaired splice-donor site at the 3' end of the exon; and (b) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell in which the exogenous regulatory sequence controls expression of a transcript comprising the construct-derived exon, the construct-derived splice-donor site, and all of the endogenous exons of the DNase I gene. 286. A homologously recombinant cell produced by the method of claim 285. 287. A cultured vertebrate cell which expresses DNase I, the genome of the cell having incorporated therein a transcription unit comprising an exogenous regulatory region, an exogenous exon, and an exogenous splice-donor site at the 3' end of the exogenous exon, wherein transcription under the control of the exogenous regulatory region produces a transcript comprising RNA corresponding to the exogenous exon, the exogenous splice-donor site, and all of the endogenous exons of an endogenous DNase I gene, wherein the RNA corresponding to the splice-donor site directs splicing to a splice-acceptor site of the transcript which corresponds to a site within the endogenous DNase I gene. 288. The cell of claim 287, wherein the splice-acceptor site of transcript corresponds to the splice-acceptor site of the second exon of the DNase I gene. 289. A method for producing DNase I comprising maintaining the cell of claim 289 under conditions appropriate for the production of DNase I under the control of the exogenous regulatory region. 290. A method for producing DNase I, comprising the steps of: (a) transfecting a cell, the chromosomal DNA of which contains an endogenous DNase I gene, with a DNA construct comprising (i) a targeting sequence homologous with a target site within or upstream of the DNase I gene; (ii) an exogenous regulatory sequence; (iii) an exon; and (iv) an unpaired splice-donor site at the 3' end of the exon, thereby creating a transfected cell; (b) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell in the genomic DNA of which (a)(ii)-(iv) are positioned upstream of the endogenous transcription initiation site of the DNase I gene, and the exogenous regulatory sequence controls expression of a transcript comprising RNA corresponding to (a)(iii)-(iv) and all of the endogenous exons of the DNase I gene; and (c) maintaining the homologously recombinant cell produced in step (b) under conditions appropriate for the production of DNase I under the control of the exogenous regulatory sequence. 291. A DNA construct which alters the expression of a .beta.-interferon gene in a cell when the DNA construct is homologously recombined with a target site within the chromosomal DNA of the cell, the construct comprising: (a) a targeting sequence homologous with a target site within or upstream of the .beta.-interferon coding region; (b) an exogenous regulatory sequence; (c) an exon; (d) a splice-donor site; (e) an intron; and (f) a splice-acceptor site, wherein, upon integration of the construct into chromosomal DNA by homologous recombination at the target site, the regulatory sequence of (b) controls expression of a transcript comprising sequence corresponding to the exon of (c), the splice-donor site of (d), the intron of (e), the splice-acceptor site of (f), and part or all of the .beta.-interferon coding region. 292. The DNA construct of claim 291, wherein the regulatory sequence comprises a promoter. 293. The DNA construct of claim 291, further comprising a selectable marker gene. 294. The DNA construct of claim 291, further comprising an amplifiable marker gene. 295. The DNA construct of claim 291, further comprising a second targeting sequence comprising DNA which hybridizes to genomic DNA within or upstream of the .beta.-interferon coding region. 296. The DNA construct of claim 291, wherein the targeting sequence is selected from the group consisting of SEQ ID NO:23, SEQ ID NO:24, fragments of SEQ ID NO:23 which selectively promote homologous recombination with genomic DNA upstream of the .beta.-interferon gene, and fragments of SEQ ID NO:23 which selectively promote homologous recombination with genomic DNA upstream of the .beta.-interferon gene. 297. The DNA construct of claim 296, wherein the targeting sequence is a fragment of SEQ ID NO:24 and is at least about 20 nucleotides. 298. The DNA construct of claim 296, wherein the targeting sequence is a fragment of SEQ ID NO:23 and is at least about 20 nucleotides. 299. An isolated DNA molecule of at least about 20 nucleotides having a sequence selected from the group consisting of SEQ ID NO:23, a fragment of SEQ ID NO:23, and a sequence which hybridizes to the complement of SEQ ID NO:23 and which selectively promotes homologous recombination with a target site upstream of a .beta.-interferon coding sequence. 300. A method of producing a homologously recombinant cell wherein the expression of a .beta.-interferon gene is altered, comprising the steps of: (a) transfecting a cell containing the .beta.-interferon gene with a DNA construct comprising (i) a targeting sequence homologous to a target site within or upstream of the .beta.-interferon coding sequence; (ii) an exogenous regulatory sequence; (iii) an exon; (iv) a splice-donor site; (v) an intron; and (vi) a splice-acceptor site, thereby producing a transfected cell; and (b) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell in which the regulatory sequence controls expression of a transcript comprising sequence corresponding to (a)(iii)-(vi) and part or all of the .beta.-interferon gene. 301. A homologously recombinant cell produced by the method of claim 300. 302. A cultured vertebrate cell which expresses .beta.-interferon, the genomic DNA of the cell comprising an exogenous regulatory region positioned to control expression of an exogenous exon, an exogenous splice-donor site, an exogenous intron, an exogenous splice-acceptor site, and part or all of an endogenous .beta.-interferon gene. 303. A method for producing .beta.-interferon, comprising the step of maintaining the cell of claim 302 under conditions appropriate for the production of .beta.-interferon. 304. A method for producing .beta.-interferon comprising the steps of: (a) transfecting a cell containing a .beta.-interferon gene with a DNA construct comprising (i) a targeting sequence homologous to a target site within or upstream of the .beta.-interferon gene; (ii) an exogenous regulatory sequence; (iii) an exon; (iv) a splice-donor site; (v) an intron; and (vi) a splice-acceptor site, thereby producing a transfected cell; (b) maintaining the transfected cell under conditions appropriate for homologous recombination in which the exogenous regulatory sequence is positioned to control expression of a transcript comprising RNA corresponding to (a)(iii)-(vi) and part or all of the .beta. interferon gene, thereby producing a homologously recombinant cell; and (c) maintaining the homologously recombinant cell produced in step (b) under conditions appropriate for the expression of .beta.-interferon under the control of the exogenous regulatory sequence. 305. A method for altering expression of a targeted gene, comprising the steps of: (a) providing a cell, the genome of which comprises (i) a targeted gene; and (ii) a target site within or upstream of the targeted gene; (b) providing a DNA construct comprising: (i) a targeting sequence homologous to the target site, and (ii) an actin promoter from a mammalian gene; (c) transfecting the cell with the DNA construct, thereby producing a transfected cell; and (d) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell the genome of which contains the construct-derived actin promoter positioned to regulate transcription of the targeted gene, the targeted gene encoding a protein other than actin; and (e) maintaining the homologously recombinant cell under conditions appropriate for transcription of the targeted gene under the control of the actin promoter. 306. A cultured vertebrate cell having incorporated therein a transcription unit comprising an exogenous actin promoter positioned to control transcription of an endogenous gene other than an actin gene in the chromosomal DNA of the cell. 307. A method of altering expression of a targeted gene, comprising the steps of: (a) providing a cell, the genome of which comprises (i) a targeted endogenous gene; and (ii) a target site within or upstream of the targeted gene; (b) providing a DNA construct comprising: (i) a targeting sequence homologous to the target site, and (ii) a collagen promoter from a mammalian gene; (c) transfecting the cell with the DNA construct, thereby producing a transfected cell; and (d) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell the genome of which contains the collagen promoter positioned to control transcription of the targeted gene, the targeted gene encoding a protein other than a collagen; and (e) maintaining the homologously recombinant cell under conditions appropriate for transcription of the targeted gene under the control of the collagen promoter. 308. A cultured vertebrate cell having incorporated therein a transcription unit, wherein the transcription unit comprising an exogenous collagen promoter positioned to control transcription of an endogenous gene other than a collagen gene in the chromosomal DNA of the cell. 309. A method of altering the expression of a targeted gene in the chromosomal DNA of a cell, comprising the steps of: (a) transfecting the cell with a DNA construct comprising: (i) a targeting sequence homologous with genomic DNA within or upstream of the coding region of the targeted gene; (ii) a non-viral promoter; (iii) an exon; and (iv) an unpaired splice-donor site at the 3' end of the exon, thereby producing a transfected cell; (b) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell the genome of which comprises the non-viral promoter, the construct-derived exon, and the construct-derived splice-donor site, in addition to all endogenous exons of the targeted gene; and (c) maintaining the homologously recombinant cell under conditions appropriate for expression of the targeted gene under the control of the non-viral promoter. 310. The method of claim 309, wherein the non-viral promoter is an actin promoter. 311. The method of claim 309, wherein the non-viral promoter is a collagen promoter. 312. A method of making a protein by altering the expression of a targeted gene that encodes the protein in a cell, comprising the steps of: (a) transfecting the cell with a DNA construct, the DNA construct comprising: (i) a targeting sequence homologous with genomic DNA within or upstream of the coding region of the targeted gene; and (ii) a non-viral promoter which is not homologous to the endogenous promoter of the targeted gene, thereby producing a transfected cell; (b) maintaining the transfected cell under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell in which the non-viral promoter is positioned to control expression of the targeted gene; and (c) maintaining the homologously recombinant cell under conditions appropriate for expression of the protein under the control of the non-viral promoter. 313. The method of claim 312, wherein the non-viral promoter is an actin promoter. 314. The method of claim 312, wherein the non-viral promoter is a collagen promoter. 315. An HT1080 cell into the genome of which is incorporated a transcription unit comprising an exogenous regulatory sequence positioned to control expression of a sequence comprising an endogenous gene, wherein the expression of the endogenous gene is altered compared to expression of the endogenous gene in an HT1080 cell lacking the transcription unit. 316. An RPMI 8226 cell into the genome of which is incorporated a transcription unit comprising an exogenous regulatory sequence positioned to control expression of a sequence comprising an endogenous gene, wherein the expression of the endogenous gene is altered compared to expression of the endogenous gene in an RPMI 8226 cell lacking the transcription unit. 317. A U-937 cell into the genome of which is incorporated a transcription unit comprising an exogenous regulatory sequence positioned to control expression of a sequence comprising an endogenous gene, wherein the expression of the endogenous gene is altered compared to expression of the endogenous gene in an RPMI 8226 cell lacking the transcription unit. 318. A WI-38VA13 subline 2R4 cell into the genome of which is incorporated a transcription unit comprising an exogenous regulatory sequence positioned to control expression of a sequence comprising an endogenous gene, wherein the expression of the endogenous gene is altered compared to expression of the endogenous gene in an WI-38VA13 subline 2R4 cell lacking the transcription unit. 319. A heterohybridoma cell into the genome of which is incorporated a transcription unit comprising an exogenous regulatory sequence positioned to control expression of a sequence comprising an endogenous gene, wherein the expression of the endogenous gene is altered compared to expression of the endogenous gene in a heterohybridoma cell lacking the transcription unit. 320. A linear DNA construct which alters the expression of a targeted gene in a cell when the DNA construct is homologously recombined with a target site within the chromosomal DNA of the cell, the DNA construct comprising: (a) a targeting sequence homologous to the target site, and (b) an exogenous regulatory sequence, wherein each end of the DNA construct has an exonuclease-generated, single-stranded overhang. 321. The construct of claim 320, wherein each of the single-stranded overhangs is a 3' overhang. 322. The construct of claim 320, wherein each of the single-stranded overhangs is a 5' overhang. 323. The construct of claim 320, wherein each of the single-stranded overhangs is between about 100 and about 1000 nucleotides in length. 324. A method of producing a protein comprising: (a) providing an HT1080 cell containing DNA comprising an exogenous regulatory region which controls expression of a sequence encoding the protein, (b) culturing the cell under conditions appropriate for production of the protein, thereby producing the protein, and (c) confirming that the protein was produced. 325. The method of claim 324, further comprising the step of: (d) isolating the protein from the cell. 326. The method of claim 325, wherein the protein is selected from the group consisting of calcitonin, insulin, insulinotropin, insulin-like growth factors, parathyroid hormone, .gamma.-interferon, nerve growth factors, TGF-.beta., tumor necrosis factor, glucagon, bone growth factor-2, bone growth factor-7, TSH-.beta., interleukin 1, interleukin 2, interleukin 3, interleukin 6, interleukin 11, interleukin 12, CSF-macrophage, immunoglobulins, catalytic antibodies, protein kinase C, superoxide dismutase, tissue plasminogen activator, urokinase, antithrombin III, DNase, tyrosine hydroxylase, blood clotting factor V, blood clotting factor VII, blood clotting factor VIII, blood clotting factor X, blood clotting factor XIII, apolipoprotein E, apolipoprotein A-I, globins, low density lipoprotein receptor, IL-2 receptor, IL-2 receptor antagonists, alpha-1 antitrypsin, immune response modifiers, and soluble CD4. 327. The method of claim 325, wherein the protein is erythropoietin. 328. The method of claim 325, wherein the protein is growth hormone. 329. The method of claim 325, wherein the protein is .alpha.-interferon. 330. The method of claim 325, wherein the protein is .beta.-interferon. 331. The method of claim 325, wherein the protein is .alpha.-galactosidase. 332. The method of claim 325, wherein the protein is glucocerebrosidase. 333. The method of claim 325, wherein the protein is FSH.beta.. 334. The method of claim 325, wherein the protein is G-CSF. 335. The method of claim 325, wherein the protein is GM-CSF. 336. The method of claim 325, wherein the protein is thrombopoietin. 337. The method of claim 325, wherein the protein is DNase I. 338. The method of claim 325, wherein the protein is blood clotting factor VIII. 339. The method of claim 325, wherein the protein is blood clotting factor IX. 340. The method of claim 324, wherein the sequence encoding the protein is an endogenous HT1080 coding sequence. 341. The method of claim 324, wherein the sequence encoding the protein is an exogenous coding sequence. 342. The method of claim 324, wherein the protein is selected from the group consisting of calcitonin, insulin, insulinotropin, insulin-like growth factors, parathyroid hormone, .alpha.-interferon, .beta.-interferon, .gamma.-interferon, nerve growth factors, TGF-.beta., tumor necrosis factor, glucagon, bone growth factor-2, bone growth factor-7, TSH-.beta., interleukin 1, interleukin 2, interleukin 3, interleukin 6, interleukin 11, interleukin 12, CSF-macrophage, CSF-granulocyte, CSF-granulocyte/macrophage, immunoglobulins, catalytic antibodies, protein kinase C, superoxide dismutase, tissue plasminogen activator, urokinase, antithrombin III, DNase, tyrosine hydroxylase, blood clotting factor V, blood clotting factor VII, blood clotting factor VIII, blood clotting factor IX, blood clotting factor X, blood clotting factor XIII, apolipoprotein E, apolipoprotein A-I, globins, low density lipoprotein receptor, IL-2 receptor, IL-2 receptor antagonists, alpha-1 antitrypsin, immune response modifiers, soluble CD4, erythropoietin, FSH.beta., growth hormone, .alpha.-galactosidase, and glucocerebrosidase. 343. The method of claim 324, wherein a DNA comprising the exogenous regulatory region and the sequence encoding the protein was introduced into a predecessor of the HT1080 cell by transfection. 344. A purified preparation of glucocerebrosidase produced by a method comprising: (a) providing a genetically engineered human cell into the genome of which is incorporated a DNA comprising an exogenous regulatory sequence positioned to control expression of a glucocerebrosidase gene, wherein said expression under the control of the exogenous regulatory sequence is altered compared to expression of glucocerebrosidase in a human cell lacking the DNA; (b) culturing the genetically engineered cell under conditions appropriate for production of glucocerebrosidase under the control of the exogenous regulatory sequence; and (c) isolating the glucocerebrosidase from the genetically engineered cell, thereby producing a purified preparation of glucocerebrosidase. 345. The preparation of claim 344, wherein the human cell is an HT1080 cell. 346. A composition comprising the preparation of claim 345 and a pharmaceutically acceptable carrier. 347. A composition comprising the preparation of claim 344 and a pharmaceutically acceptable carrier. 348. A fusion protein comprising amino acids 1-3 of human growth hormone signal peptide and amino acids 5-165 of human erythropoietin. 349. The fusion protein of claim 348, consisting of amino acids 1-3 of the human growth hormone signal peptide and amino acids 5-165 of human erythropoietin. 350. A fusion protein consisting of a first sequence consisting of amino acids 1-3 of human growth hormone signal peptide fused to a second sequence which is not part of human growth hormone signal peptide. 351. The fusion protein of claim 350, wherein the second sequence consists of part of erythropoietin. 352. A cultured vertebrate cell having incorporated therein a transcription unit, wherein the transcription unit comprises an exogenous non-viral promoter positioned to control expression of an endogenous gene in the chromosomal DNA of the cell, provided that the exogenous non-viral promoter alters expression of the endogenous gene compared to expression under the control of the endogenous promoter of the endogenous gene. 353. A cultured vertebrate cell which expresses a GM-CSF, the genome of said cell having incorporated therein a transcription unit comprising an exogenous regulatory sequence, an exogenous exon, and an exogenous splice-donor site, wherein transcription under the control of the exogenous regulatory sequence produces a transcript comprising RNA corresponding to the exogenous exon, the exogenous splice-donor site, and all of the endogenous exons of an endogenous GM-CSF gene, wherein the RNA corresponding to the splice-donor site directs splicing to a splice-acceptor site of the transcript which corresponds to a site within the endogenous GM-CSF gene. 354. The cell of claim 353, wherein the splice-acceptor site of the transcript corresponds to the splice-acceptor site of the second exon of the GM-CSF gene. 355. A cultured vertebrate cell which expresses FSH.beta., the genome of the cell having incorporated therein a transcription unit comprising an exogenous regulatory region, an exogenous exon, and an exogenous splice-donor site at the 3' end of the exogenous exon, wherein transcription under the control of the exogenous regulatory region produces a transcript comprising RNA corresponding to the exogenous exon, the exogenous splice-donor site, and all of the endogenous exons of an endogenous FSH.beta. gene, wherein the RNA corresponding to the splice-donor site directs splicing to a splice-acceptor site of the transcript which corresponds to a site within the endogenous FSH.beta. gene. 356. The cell of claim 355 wherein the exogenous splice-donor site is operatively linked to the endogenous splice-acceptor site of the second exon of the FSH.beta. gene.

Details for Patent 6,270,989

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Microbix Biosystems Inc.	KINLYTIC	urokinase	For Injection	021846	01/16/1978		2018-08-07
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2018-08-07
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2018-08-07