Cells and methods for the generation of transgenic pigs

• US 6,271,436 B1
• Filed: 10/10/1997
• Issued: 08/07/2001
• Est. Priority Date: 10/11/1996
• Status: Expired due to Fees

• Current Assignee: Texas A&M University System
• Original Assignee: Texas A&M University System
• Inventors: Bazer, Fuller W., Piedrahita, Jorge A.
• Primary Examiner(s): Martin, Jill D.
• Application Number:
  US08/949,155
• Time in Patent Office: 1,397 Days
• Field of Search: 435/325, 435/383, 435/384, 435/455, 435/463, 435/461, 435/459, 435/462, 800/13, 800/14, 800/15, 800/16, 800/17, 800/21, 800/22, 800/24, 800/25, 800/18
• US Class Current: 800/21
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: C12N 2500/24 Iron; Fe chelators; Transferrin
• CPC Class Codes: C12N 2500/32 Amino acids
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/125 Stem cell factor [SCF], c-kit ligand [KL]
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2517/04 Cells produced using nuclear transfer
• CPC Class Codes: C12N 2517/10 Conditioning of cells for in vitro fecondation or nuclear transfer
• CPC Class Codes: C12N 5/0611 Primordial germ cells, e.g. embryonic germ cells [EG]

Abstract

Disclosed are methods for the isolation of primordial germ cells, culturing these cells to produce primordial germ cell-derived cell lines, methods for transforming both the primordial germ cells and the cultured cell lines, and using these transformed cells and cell lines to generate transgenic animals. The efficiency at which transgenic animals are generated by the present invention is greatly increased, thereby allowing the use of homologous recombination in producing transgenic non-rodent animal species.

Claims

1. A method of growing porcine primordial germ cells, comprising growing a cell culture comprising porcine primordial germ cells from an embryo of a pig on feeder cells for a time sufficient to obtain undifferentiated porcine primordial germ cells, said feeder cells at a density of between about 2.5×

10⁵ cells/cm² and about 10⁶ cells/cm², in a culture medium comprising an effective amount of basic fibroblast growth factor.

2. The method of claim 1, wherein said cell culture comprising porcine primordial germ cells is isolated from in embryo of said pig.

3. The method of claim 2, wherein said cell culture comprising porcine primordial germ cells is isolated by a process comprising the steps of:

a) removing the genital ridge from said embryo;

b) disrupting said genital ridge in an appropriate solution, thereby releasing porcine primordial germ cells; and

c) collecting said porcine primordial germ cells, to provide said cell culture comprising porcine primordial germ cells.

4. The method of claim 1, wherein said porcine primordial germ cells comprise at least a first exogenous DNA segment.

5. The method of claim 1, wherein said feeder cells are STO cells.

6. The method of claim 1, wherein said culture medium comprises basic fibroblast growth factor at a concentration of between about 5 ng/ml and about 100 μ

g/ml.

7. The method of claim 1, wherein said culture medium further comprises an effective amount of uteroferrin.

8. The method of claim 7, wherein said culture medium comprises uteroferrin at a concentration of between about 1 ng/ml and about 100 μ

g/ml.

9. The method of claim 1, wherein said culture medium further comprises an effective amount of α

2-macroglobulin.

10. The method of claim 9, wherein said culture medium comprises α

2-macroglobulin at a concentration of between about 10 ng/ml and about 10 μ

g/ml.

11. The method of claim 1, wherein said culture medium further comprises an effective amount of leukemia inhibitory factor.

12. The method of claim 11, wherein said culture medium comprises leukemia inhibitory factor at a concentration of between about 5 ng/ml and about 100 μ

g/ml.

13. The method of claim 1, wherein said culture medium further comprises an effective amount of soluble stem cell factor.

14. The method of claim 13, wherein said culture medium comprises soluble stem cell factor at a concentration of between about 1 ng/ml and about 100 μ

g/ml.

15. The method of claim 1, wherein said culture medium further comprises an effective amount of amino acids non-essential with respect to said pig.

16. The method of claim 15, wherein said culture medium comprises amino acids non-essential to said pig at a concentration of between about 10 nM and about 250 nM.

17. The method of claim 1, wherein said culture medium further comprises an effective amount of L-glutamine.

18. The method of claim 17, wherein said culture medium comprises L-glutamine at a concentration of between about 0.1 mM and about 50 mM.

19. The method of claim 1, wherein said culture medium further comprises an effective amount of β

-mercaptoethanol.

20. The method of claim 19, wherein said culture medium comprises β

-mercaptoethanol at a concentration of between about 1 μ

M and about 1 mM.

21. The method of claim 1, wherein said culture medium comprises Dulbecco'"'"'s modified Eagle'"'"'s media or Ham'"'"'s F10 media.

22. The method of claim 1, wherein said culture medium comprises Dulbecco'"'"'s modified Eagle'"'"'s media at about 50% volume/volume and Ham'"'"'s F10 media at about 50% volume/volume.

23. The method of claim 1, wherein said culture medium comprises an effective amount of basic fibroblast growth factor and an effective amount of at least one of uteroferrin, α

2-macroglobulin, leukemia inhibitory factor, soluble stem cell factor, amino acids non-essential with respect to said pig, L-glutamine or α

-mercaptoethanol.

24. The method of claim 23, wherein said culture medium comprises an effective amount of basic fibroblast growth factor and a combined effective amount of at least two of uteroferrin, α

2-macroglobulin, leukemia inhibitory factor, soluble stem cell factor, amino acids non-essential with respect to said pig, L-glutamine or β

-mercaptoethanol.

25. The method of claim 24, wherein said culture medium comprises an effective amount of basic fibroblast growth factor and a combined effective amount of at least three of uteroferrin, α

2-macroglobulin, leukemia inhibitory factor, soluble stem cell factor, amino acids non-essential with respect to said pig, L-glutamine or β

-mercaptoethanol.

26. The method of claim 25, wherein said culture medium comprises an effective amount of basic fibroblast growth factor and a combined effective amount of uteroferrin, α

2-macroglobulin and leukemia inhibitory factor.

27. The method of claim 26, wherein said culture medium comprises basic fibroblast growth factor at a concentration of between about 5 ng/ml and about 100 μ

g/ml, uteroferrin at a concentration of between about 1 ng/ml and about 100 μ

g/ml, α

2-macroglobulin at a concentration of between about 10 ng/ml and about 10 μ

g/ml and leukemia inhibitory factor at a concentration of between about 5 ng/ml and about 100 μ

g/ml.

28. The method of claim 1, wherein said culture medium comprises:

a) between about 5 ng/ml and about 100 μ

g/ml of basic fibroblast growth factor;

b) between about 1 ng/ml and about 100 μ

g/ml of uteroferrin;

c) between about 10 ng/ml and about 10 μ

g/ml of α

2-macroglobulin;

d) between about 5 ng/ml and about 100 μ

g/ml of leukemia inhibitory factor;

e) between about 1 ng/ml and about 100 μ

g/ml of soluble stem cell factor;

f) between about 10 nM and about 250 nM of amino acids nonessential with respect to said pig;

g) between about 0.1 mM and about 50 mM of L-glutamine;

h) between about 1 μ

M and about 1 mM of β

-mercaptoethanol;

i) about 50% volume/volume of Dulbecco'"'"'s modified Eagle'"'"'s media; and

j) about 50% volume/volume of Ham'"'"'s F10 media.

29. The method of claim 1, wherein the porcine primordial germ cells are maintained in an undifferentiated state for between about 2 passages and about 14 passages.

30. A method of growing porcine primordial germ cells, comprising growing a cell culture comprising porcine primordial germ cells from an embryo of a pig on STO feeder cells for a time sufficient to obtain undifferentiated porcine primordial germ cells, said STO feeder cells at a density of between about 2.5×

10⁵ cells/cm² and about 10⁶ cells/cm², in a culture medium comprising an effective amount of basic fibroblast growth factor.

31. A method of growing porcine primordial germ cells, comprising growing a cell culture comprising porcine primordial germ cells from an embryo of a pig on feeder cells for a time sufficient to obtain undifferentiated porcine primordial germ cells, said feeder cells at a density of between about 2.5×

10⁵ cells/cm² and about 10⁶ cells/cm², in a culture medium comprising an effective amount of basic fibroblast growth factor, said culture medium including no exogenously added soluble stem cell factor.

32. A method of growing porcine primordial germ cells, comprising growing a cell culture comprising porcine primordial germ cells from an embryo of a pig on feeder cells for a time sufficient to obtain undifferentiated porcine primordial germ cells, said feeder cells at a density of between about 2.5×

10⁵ cells/cm² and about 10⁶ cells/cm², in a culture medium comprising an effective amount of basic fibroblast growth factor, said culture medium including no exogenously added leukemia inhibitory factor.

33. A method of growing porcine primordial germ cells, comprising growing a cell culture comprising porcine primordial germ cells from an embryo of a pig on feeder cells for a time sufficient to obtain undifferentiated porcine primordial germ cells, said feeder cells at a density of between about 2.5×

10⁵ cells/cm² and about 10⁶ cells/cm², in a culture medium comprising an effective amount of basic fibroblast growth factor, said culture medium including no exogenously added soluble stem cell factor or leukemia inhibitory factor.

34. A method of growing porcine primordial germ cells, comprising growing a cell culture comprising porcine primordial germ cells from an embryo of a pig on feeder cells other than S1/S1⁴ or S1-m220 for a time sufficient to obtain undifferentiated porcine primordial germ cells, said feeder cells at a density of between about 2.5×

10⁵ cells/cm² and about 10⁶ cells/cm², in a culture medium comprising an effective amount of basic fibroblast growth factor.

35. A method of growing porcine primordial germ cells, comprising growing a cell culture comprising porcine primordial germ cells from an embryo of a pig on feeder cells other than S1/S1⁴ or S1-m220 for a time sufficient to obtain undifferentiated porcine primordial germ cells, said feeder cells at a density of between about 2.5×

10⁵ cells/cm² and about 10⁶ cells/cm², in a culture medium comprising an effective amount of basic fibroblast growth factor, said culture medium including no exogenously added soluble stem cell factor or leukemia inhibitory factor.

36. A method of preparing a porcine primordial germ cell-derived cell line, comprising:

a) plating a cell culture comprising porcine primordial germ cells on feeder cells, said feeder cells at a density of between about 2.5×

10 cells/cm² and about 10⁶ cells/cm², in a culture medium comprising an effective amount of basic fibroblast growth factor; and

b) culturing the plated porcine primordial germ cells for a period of time effective to provide a porcine primordial germ cell-derived cell line.

37. A method of preparing porcine primordial germ cells that contain a selected DNA segment, comprising:

a) introducing said selected DNA segment into a composition comprising porcine primordial germ cells to obtain candidate porcine primordial germ cells that contain said selected DNA segment; and

b) plating said candidate porcine primordial germ cells that contain said selected DNA segment on feeder cells, said feeder cells at a density of between about 2.5×

10⁵ cells/cm² and about 10⁶ cells/cm², in a culture medium comprising an effective amount of basic fibroblast growth factor, to obtain said porcine primordial germ cells that contain said selected DNA segment.

38. The method of claim 37, further comprising culturing said porcine primordial germ cells that contain said selected DNA segment for between about 2 and about 14 passages.

39. The method of claim 37, wherein said selected DNA segment is introduced into said porcine primordial germ cells by electroporation, particle bombardment or viral transformation.

40. The method of claim 37, wherein said selected DNA segment comprises at least a first coding region encoding a selected protein.

41. The method of claim 40, wherein said first coding region encodes a selected marker protein.

42. The method of claim 41, wherein said first coding region encodes green fluorescent protein.

43. The method of claim 40, wherein said first coding region encodes a protein, wherein said protein is an interleukin, collagen, interferon, blood protein, hormone, growth factor, cytokine, enzyme, receptor, binding protein, immune system protein, antigen, muscle protein or oncogene receptor.

44. The method of claim 43, wherein said first coding region encodes a protein, wherein said protein is a SREHP, GP63, actinobacillus, pleuropneumoniae, pseudomonas aeruynosa, OprF, myelin basic protein, insulin, hCD59, DAF (CD55), factor IX, urokinase, α

-antitrypsin, tissue plasminogen activator, protein C, activin, adenosine dearinase, angiotensinogen I, antithrombin III, alpha I antitrypsin, apolipoprotein A-I, apolipoprotein A-II, apolipoprotein C-I, apolipoprotein C-II, apolipoprotein C-III, apolipoprotein E, atrial natriuretic factor, chorionic gonadotropin, alpha chain, beta chain, pro (rennin) chymosin, factor B complement, complement C2, complement C3, complement C4, complement C9, corticotropin releasing factor, epidermal growth factor, c-erb B, epoxide dehydratase, erythropoietin, C1 esterase inhibitor, factor VIII, factor IX, Christmas factor, factor X, fibrinogen A alpha, gamma B beta, gastrin releasing peptide, prepro glucagon, growth hormone, RF growth hormone, somatocrinin, hemopexin, inhibin, prepro insulin, insulin-like growth factor I, insulin-like growth factor II, alpha interferon, multiple leukocyte, fibroblast beta interferon, gamma interferon, interleukin-1, T-cell interleukin-2, growth factor, interleukin-3, two forms kininogen, beta subunit leuteinizing hormone, leuteinizing hormone, releasing hormone, lymphotoxin, mast cell growth factor, beta subunit nerve growth factor, PGDF c-sis oncogene, chain A, pancreatic polypeptide, icosapeptide, parathyroid hormone, prepro plasminogen, plasminogen activator, prolactin, proopiomelanocortin, protein C, prothromnbin, relaxin, prepro renin, somatostatin, prepro tachykinin, substance P, substance K, urokinase or prepro vasoactive intestinal peptide protein.

45. The method of claim 40, wherein said selected DNA segment further comprises at least a second coding region encoding a selected protein.

46. The method of claim 45, wherein said first coding region encodes a selected protein and said second coding region encodes a selected marker protein.

47. The method of claim 37, wherein said DNA segment is operatively positioned under the control of a promoter.

48. The method of claim 47, wherein said promoter is a CMV, Oct-4 or pgk promoter.

49. The method of claim 41, wherein said selected DNA segment is operatively positioned in reverse orientation under the control of a promoter, wherein said promoter directs the expression of an antisense product.

50. The method of claim 37, wherein said DNA segment further comprises two selected DNA regions that flank said DNA segment, thereby directing the homologous recombination of said DNA segment into genomic DNA of said porcine primordial germ cells.

51. The method of claim 50, wherein said selected DNA regions comprise porcine genomic DNA.

52. The method of claim 51, wherein said selected DNA regions comprise genomic DNA from the Oct-4 gene.

53. The method of claim 50, wherein said DNA segment further comprises two selected DNA sequences that flank said DNA segment, thereby directing excision of said DNA segment under appropriate conditions.

54. The method of claim 53, wherein said selected DNA sequences are loxP sites.

55. A method of producing a transgenic pig, comprising:

a) introducing a selected DNA segment into a cell culture comprising porcine primordial germ cells to obtain candidate porcine primordial germ cells that contain said selected DNA segment;

b) plating said candidate porcine primordial germ cells that contain said selected DNA segment on feeder cells, said feeder cells at a density of between about 2.5×

10⁵ cells/cm² and about 10⁶ cells/cm², in a culture medium comprising an effective amount of basic fibroblast growth factor, to obtain undifferentiated porcine primordial germ cells that contain said selected DNA segment; and

c) generating a transgenic pig from said undifferentiated porcine primordial germ cells that contain said selected DNA segment, wherein said selected DNA segment is contained and expressed in somatic and germ cells of said transgenic pig.

56. The method of claim 55, wherein said cell culture comprising porcine primordial germ cells contains undifferentiated cultured cells from an undifferentiated porcine primordial germ cell-derived cell line.

57. The method of claim 55, wherein said transgenic pig is generated by a method comprising:

(a) injecting said undifferentiated porcine primordial germ cells that contain said selected DNA segment into a blastocyst from a pig;

(b) transferring said blastocyst into a synchronized recipient female pig to produce a pregnant pig; and

(c) allowing gestation in said pregnant pig to proceed for a period of time sufficient to allow the development of a viable transgenic pig.

58. The method of claim 55, wherein said transgenic pig is generated by a method comprising;

(a) isolating a nucleus from said undifferentiated porcine primordial germ cells that contain said selected DNA segment and injecting said nucleus into an enucleated oocyte from a pig;

(b) transferring said oocyte into a synchronized recipient female pig to produce a pregnant pig; and

(c) allowing gestation in said pregnant pig to proceed for a period of time sufficient to allow the development of a viable transgenic pig.

59. The method of claim 55, wherein said transgenic pig is generated by a method comprising:

(a) aggregating said undifferentiated porcine primordial germ cells that contain said selected DNA segment with an early stage embryo of a pig;

(b) transferring said embryo into a synchronized recipient female pig to produce a pregnant pig; and

(c) allowing gestation in said pregnant pig to proceed for a period of time sufficient to allow the development of a viable transgenic pig.

60. The method of claim 55, wherein said selected DNA segment comprises at least a first coding region that encodes a protein selected from the group consisting of an interleukin, collagen, interferon, blood protein, hormone, growth factor, cytokine, enzyme, receptor, binding protein, immune system protein, antigen, muscle protein and an oncogene receptor.

61. The method of claim 60, wherein said selected DNA segment comprises at least a first coding region that encodes a protein selected from the group consisting of SREHP, GP63, actinobacillus, pleuropneumoniae, pseudomonas aeruynosa, OprF, myelin basic protein, insulin, hCD59, DAF (CD55), factor IX, urokinase, α

-antitrypsin, tissue plasminogen activator, protein C, activin, adenosine deaminase, angiotensinogen 1, antithrombin III, alpha I antitrypsin, apolipoprotein A-I, apolipoprotein A-II, apolipoprotein C-I, apolipoprotein C-II, apolipoprotein C-III, apolipoprotein E, atrial natriuretic factor, chorionic gonadotropin, alpha chain, beta chain, pro (rennin) chymosir, factor B complement, complement C2, complement C3, complement C4, complement C9, corticotropin releasing factor, epidermal growtih factor, c-erb B, epoxide dehydratase, erythropoietin, C1 esterase inhibitor, factor VIII, factor IX, Christmas factor, factor X, fibrinogen A alpha, gamma B beta, gastrin releasing peptide, prepro glucagon, growth hormone, RF growth hormone, somatocrinin, hemopexin, inhibin, prepro insulin, insulin-like growth factor I, insulin-like growth factor II, alpha interferon, multiple leukocyte, fibroblast beta interferon, gamma interferon, interleukin-1, T-cell interleukin-2, growth factor, interleukin-3, two forms kininogen, beta subunit leuteinizing hormone, leuteinizing hormone, releasing hormone, lymphotoxin, mast cell growth factor, beta subunit nerve growth factor, PGDF c-sis oncogene, chain A. pancreatic polypeptide, icosapeptide, parathyroid hormone, prepro plasminogen, plasminogen activator, prolactini, proopiomelanocortin, protein C, prothrombin, relaxin, prepro renin, somatostatin, prepro tachykinin, substance P, substance K, urokinase and prepro vasoactive intestinal peptide protein.

62. A cell culture comprising:

(a) porcine primordial germ cells;

(b) feeder cells sufficient to achieve a density of between about 2.5×

10⁵ cells/cm² and about 10⁶ feeder cells/cm²; and

(c) basic fibroblast growth factor in an amount effective to promote the growth and continued proliferation of said porcine primordial germ cells.

63. The cell culture of claim 62, wherein said porcine primordial germ cells comprise at least a first exogenous DNA segment.

64. The cell culture of claim 62, wherein said feeder cells are STO cells.

65. The cell culture of claim 62, further comprising one or more of uteroferrin, α

2-macroglobulin, leukemia inhibitory factor, soluble stem cell factor, amino acids non-essential with respect to said porcine primordial germ cells, L-glutamine, β

-mercaptoethanol, Dulbecco'"'"'s modified Eagle'"'"'s media or Ham'"'"'s F10 media in an amount effective to promote the growth and continued proliferation of said porcine primordial germ cells.

66. A kit comprising, in suitable container means:

(a) porcine primordial germ cells;

(b) feeder cells sufficient to achieve a density of between about 2.5×

10⁵ cells/cm² and about 10⁶ feeder cells/cm²; and

(c) basic fibroblast growth factor in an amount effective to promote the growth and continued proliferation of said porcine primordial germ cells.

67. A method of preparing a porcine blastocyst that contains a selected DNA segment, comprising:

a) introducing said selected DNA segment into a cell culture comprising porcine primordial germ cells to obtain candidate porcine primordial germ cells that contain said selected DNA segment;

b) plating said candidate porcine primordial germ cells that contain said selected DNA segment on feeder cells, said feeder cells at a density of between about 2.5×

10⁵ cells/cm² and about 10⁶ cells/cm², in a culture medium comprising an effective amount of basic fibroblast growth factor, to obtain undifferentiated porcine primordial germ cells that contain said selected DNA segment; and

c) injecting said undifferentiated porcine primordial germ cells that contain said selected DNA segment into a porcine blastocyst, thereby preparing a porcine blastocyst that contains said selected DNA segment.

68. A method of preparing a porcine oocyte that contains a selected DNA segment, comprising:

a) introducing said selected DNA segment into a cell culture comprising porcine primordial germ cells to obtain candidate porcine primordial germ cells that contain said selected DNA segment;

b) plating said candidate porcine primordial germ cells that contain said selected DNA segment on feeder cells, said feeder cells at a density of between about 2.5×

10⁵ cells/cm² and about 10⁶ cells/cm², in a culture medium comprising an effective amount of basic fibroblast growth factor, to obtain undifferentiated porcine primordial germ cells that contain said selected DNA segment;

c) isolating a nucleus from said undifferentiated porcine primordial germ cells that contain said selected DNA segment; and

d) injecting said nucleus into an enucleated porcine oocyte, thereby preparing a porcine oocyte that contains said selected L)NA segment.

69. A method of preparing an early stage porcine embryo that contains a selected DNA segment, comprising:

a) introducing a selected DNA segment into a cell culture comprising porcine primordial germ cells to obtain candidate porcine primordial germ cells that contain said selected DNA segment;

b) plating said candidate porcine primordial germ cells that contain said selected DNA segment on feeder cells, said feeder cells at a density of between about 2.5×

10⁵ cells/cm² and about 10⁶ cell/cm², in a culture medium comprising an effective amount of basic fibroblast growth factor, to obtain undifferentiated porcine primordial germ cells that contain said selected DNA segment; and

c) aggregating said undifferentiated porcine primordial germ cells that contain said selected DNA segment with an early stage porcine embryo, thereby preparing an early stage porcine embryo that contains said selected DNA segment.