Injection of caprine sperm factor (cSF), phospholipase C zeta (PLCzeta) and adenophostin A as alternative methods of activation during nuclear transfer in the caprine species

• US 20060168671A1
• Filed: 01/21/2005
• Published: 07/27/2006
• Est. Priority Date: 01/21/2005
• Status: Abandoned Application

• Current Assignee: GTC Biotherapeutics, Inc.
• Original Assignee: GTC Biotherapeutics, Inc.
• Inventors: Gavin, William G., Jellerette, Teru
• Application Number:
  US11/040,271
• Publication Number:
  US 20060168671A1
• Time in Patent Office: Days
• US Class Current: 800/14
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2227/102 Caprine
• CPC Class Codes: C12N 15/873 Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos

Abstract

It has been found that caprine sperm factor, alone or in combination with adenophostin A, produces the Ca²⁺ oscillations characteristic of egg activation seen with fertilization. The introduction of caprine sperm factor and adenophostin A to eggs to produce reconstructed embryos has been optimized.

Claims

1. A method for inducing caprine egg activation, said method comprising introducing a composition comprising isolated caprine sperm factor into one or more reconstructed caprine eggs.

2. The method of claim 1, wherein said donor cell or donor cell nucleus is from an ungulate selected from the group consisting of bovine, ovine, porcine, equine, caprine and buffalo.

3. The resultant offspring of the method of claim 1.

4. A method for inducing caprine egg activation, said method comprising introducing a composition comprising adenophostin A into one or more reconstructed caprine eggs.

5. A method for inducing caprine egg activation, said method comprising introducing a composition comprising isolated caprine sperm factor and adenophostin A into one or more reconstructed caprine eggs.

6. A method for inducing caprine egg activation, said method comprising introducing, simultaneously with nuclear transfer, a composition comprising isolated caprine sperm factor into one or more enucleated caprine eggs.

7. A method for inducing caprine egg activation, said method comprising introducing, simultaneously with nuclear transfer, a composition comprising adenophostin A into one or more enucleated caprine eggs.

8. A method for inducing caprine egg activation, said method comprising introducing, simultaneously with nuclear transfer, a composition comprising isolated caprine sperm factor and adenophostin A into one or more enucleated caprine eggs.

9. A method for inducing caprine egg activation, said method comprising introducing, preceding nuclear transfer, a composition comprising isolated caprine sperm factor into one or more enucleated caprine eggs.

10. A method for inducing caprine egg activation, said method comprising introducing, preceding nuclear transfer, a composition comprising adenophostin A into one or more enucleated caprine eggs.

11. The method of claim 10, wherein said donor cell or donor cell nucleus is from an ungulate selected from the group consisting of bovine, ovine, porcine, equine, caprine and buffalo.

12. A method for inducing caprine egg activation, said method comprising introducing, preceding nuclear transfer, a composition comprising isolated caprine sperm factor and adenophostin A into one or more enucleated caprine eggs.

13. A method for cloning a non-human mammal through a nuclear transfer process comprising:

(i) obtaining desired differentiated mammalian cells to be used as a source of donor nuclei;

(ii) obtaining at least one oocyte from a mammal of the same species as the cells which are the source of donor nuclei;

(iii) enucleating said at least one oocyte;

(iv) transferring the desired differentiated cell or cell nucleus into the enucleated oocyte;

(v) simultaneously fusing and activating the cell couplet to form a transgenic embryo in the presence of caprine sperm factor;

(vi) culturing said activated transgenic embryo until greater than the 2-cell developmental stage; and

(vii) transferring said transgenic embryo into a host mammal such that the embryo develops into a fetus.

14. The method of claim 13, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from mesoderm.

15. The method of claim 13, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from endoderm.

16. The method of claim 13, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from ectoderm.

17. The method of claim 13, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from fetal somatic tissue.

18. The method of claim 13, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from fetal somatic cells.

19. The method of claim 13, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from a fibroblast.

20. The method of claim 13, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from an ungulate.

21. The method of claim 13, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from an adult non-human mammalian somatic cell.

22. The method of claim 13, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is selected from the group consisting of epithelial cells, neural cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, B-lymphocytes, T-lymphocytes, erythrocytes, macrophages, monocytes, fibroblasts, and muscle cells.

23. The method of claim 13, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from an organ selected from the group consisting of skin, lung, pancreas, liver, stomach, intestine, heart, reproductive organ, bladder, kidney and urethra.

24. The method of claim 13, wherein said at least one oocyte is matured in vivo prior to enucleation.

25. The method of claim 13, wherein said at least one oocyte is matured in vitro prior to enucleation.

26. The method of claim 13, wherein said non-human mammal is a rodent.

27. The method of claim 13, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is a non-quiescent somatic cell or a nucleus isolated from said non-quiescent somatic cell.

28. The method of claim 13, wherein the fetus develops into an offspring.

29. The method of claim 13, wherein said at least one oocyte is enucleated about 10 to 60 hours after initiation of in vitro maturation.

30. The method of claim 13, wherein a desired gene is inserted, removed or modified in said differentiated mammalian cell or cell nucleus prior to insertion of said differentiated mammalian cell or cell nucleus into said enucleated oocyte.

31. The resultant offspring of the method of claim 30.

32. The method of claims 13, wherein cytocholasin-B is used in the cloning protocol.

33. The method of claim 13, wherein cytocholasin-B is not used in the cloning protocol.

34. A method for cloning a non-human mammal through a nuclear transfer process comprising:

(i) obtaining desired differentiated mammalian cells to be used as a source of donor nuclei;

(ii) obtaining at least one oocyte from a mammal of the same species as the cells which are the source of donor nuclei;

(iii) enucleating said at least one oocyte;

(iv) transferring the desired differentiated cell or cell nucleus into the enucleated oocyte;

(v) simultaneously fusing and activating the cell couplet to form a transgenic embryo in the presence of Adenophostin A;

(vi) culturing said activated transgenic embryo until greater than the 2-cell developmental stage; and

(vii) transferring said transgenic embryo into a host mammal such that the embryo develops into a fetus.

35. The method of claim 34, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from mesoderm.

36. The method of claim 34, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from endoderm.

37. The method of claim 34, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from ectoderm.

38. The method of claim 34, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from fetal somatic tissue.

39. The method of claim 34, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from fetal somatic cells.

40. The method of claim 34, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from a fibroblast.

41. The method of claim 34, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from an ungulate.

42. The method of claim 34, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from an adult non-human mammalian somatic cell.

43. The method of claim 34, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is selected from the group consisting of epithelial cells, neural cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, B-lymphocytes, T-lymphocytes, erythrocytes, macrophages, monocytes, fibroblasts, and muscle cells.

44. The method of claim 34, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from an organ selected from the group consisting of skin, lung, pancreas, liver, stomach, intestine, heart, reproductive organ, bladder, kidney and urethra.

45. The method of claim 34, wherein said at least one oocyte is matured in vivo prior to enucleation.

46. The method of claim 34, wherein said at least one oocyte is matured in vitro prior to enucleation.

47. The method of claim 34, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is a non-quiescent somatic cell or a nucleus isolated from said non-quiescent somatic cell.

48. The method of claim 34, wherein said non-human mammal is a rodent.

49. The method of claim 34, wherein the fetus develops into an offspring.

50. The method of claim 34, wherein a desired gene is inserted, removed or modified in said differentiated mammalian cell or cell nucleus prior to insertion of said differentiated mammalian cell or cell nucleus into said enucleated oocyte.

51. The method of claim 34, wherein said at least one oocyte is enucleated about 10 to 60 hours after initiation of in vitro maturation.

52. The resultant offspring of the method of claim 51.

53. The method of claim 34, wherein cytocholasin-B is not used in the cloning protocol.

54. The method of claims 34, wherein cytocholasin-B is used in the cloning protocol.

55. A method for cloning a non-human mammal through a nuclear transfer process comprising:

(i) obtaining desired differentiated mammalian cells to be used as a source of donor nuclei;

(ii) obtaining at least one oocyte from a mammal of the same species as the cells which are the source of donor nuclei;

(iii) enucleating said at least one oocyte;

(iv) transferring the desired differentiated cell or cell nucleus into the enucleated oocyte;

(v) simultaneously fusing and activating the cell couplet to form a transgenic embryo in the presence of Adenophostin A and caprine sperm factor;

(vi) culturing said activated transgenic embryo until greater than the 2-cell developmental stage; and

(vii) transferring said transgenic embryo into a host mammal such that the embryo develops into a fetus.

56. The method of claim 55, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from mesoderm.

57. The method of claim 55, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from endoderm.

58. The method of claim 55, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from ectoderm.

59. The method of claim 55, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from fetal somatic tissue.

60. The method of claim 55, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from fetal somatic cells.

61. The method of claim 55, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from a fibroblast.

62. The method of claim 55, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from an ungulate.

63. The method of claim 55, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from an adult non-human mammalian somatic cell.

64. The method of claim 55, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is selected from the group consisting of epithelial cells, neural cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, B-lymphocytes, T-lymphocytes, erythrocytes, macrophages, monocytes, fibroblasts, and muscle cells.

65. The method of claim 55, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from an organ selected from the group consisting of skin, lung, pancreas, liver, stomach, intestine, heart, reproductive organ, bladder, kidney and urethra.

66. The method of claim 55, wherein said at least one oocyte is matured in vivo prior to enucleation.

67. The method of claim 55, wherein said at least one oocyte is matured in vitro prior to enucleation.

68. The method of claim 55, wherein said non-human mammal is a rodent.

69. The method of claim 55, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is a non-quiescent somatic cell or a nucleus isolated from said non-quiescent somatic cell.

70. The method of claim 55, wherein the fetus develops into an offspring.

71. The method of claim 55, wherein said at least one oocyte is enucleated about 10 to 60 hours after initiation of in vitro maturation.

72. The method of claim 55, wherein a desired gene is inserted, removed or modified in said differentiated mammalian cell or cell nucleus prior to insertion of said differentiated mammalian cell or cell nucleus into said enucleated oocyte.

73. The resultant offspring of the methods of claim 72.

74. The method of claim 55, wherein cytocholasin-B is used in the cloning protocol.

75. The method of claim 55, wherein cytocholasin-B is not used in the cloning protocol.

76. A method for cloning a non-human mammal through a nuclear transfer process comprising:

(i) obtaining desired differentiated mammalian cells to be used as a source of donor nuclei;

(ii) obtaining at least one oocyte from a mammal of the same species as the cells which are the source of donor nuclei;

(iii) enucleating said at least one oocyte;

(iv) transferring the desired differentiated cell or cell nucleus into the enucleated oocyte;

(v) simultaneously fusing and activating the cell couplet to form a transgenic embryo in the presence of PLCζ

;

(vi) culturing said activated transgenic embryo until greater than the 2-cell developmental stage; and

(vii) transferring said transgenic embryo into a host mammal such that the embryo develops into a fetus.

77. The method of claim 76, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from mesoderm.

78. The method of claim 76, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from endoderm.

79. The method of claim 76, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from ectoderm.

80. The method of claim 76, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from fetal somatic tissue.

81. The method of claim 76, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from fetal somatic cells.

82. The method of claim 76, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from a fibroblast.

83. The method of claim 76, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from an ungulate.

84. The method of claim 76, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from an adult non-human mammalian somatic cell.

85. The method of claim 76, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is selected from the group consisting of epithelial cells, neural cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, B-lymphocytes, T-lymphocytes, erythrocytes, macrophages, monocytes, fibroblasts, and muscle cells.

86. The method of claim 76, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from an organ selected from the group consisting of skin, lung, pancreas, liver, stomach, intestine, heart, reproductive organ, bladder, kidney and urethra.

87. The method of claim 76, wherein said at least one oocyte is matured in vivo prior to enucleation.

88. The method of claim 76, wherein said at least one oocyte is matured in vitro prior to enucleation.

89. The method of claim 76, wherein said non-human mammal is a rodent.

90. The method of claim 76, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is a non-quiescent somatic cell or a nucleus isolated from said non-quiescent somatic cell.

91. The method of claim 76, wherein the fetus develops into an offspring.

92. The method of claim 76, wherein said at least one oocyte is enucleated about 10 to 60 hours after initiation of in vitro maturation.

93. The method of claim 76, wherein a desired gene is inserted, removed or modified in said differentiated mammalian cell or cell nucleus prior to insertion of said differentiated mammalian cell or cell nucleus into said enucleated oocyte.

94. The resultant offspring of the methods of claim 93.

95. The method of claim 76, wherein cytocholasin-B is used in the cloning protocol.

96. The method of claim 76, wherein cytocholasin-B is not used in the cloning protocol.

97. A method for cloning a non-human mammal through a nuclear transfer process comprising:

(i) obtaining desired differentiated mammalian cells to be used as a source of donor nuclei;

(ii) obtaining at least one oocyte from a mammal of the same species as the cells which are the source of donor nuclei;

(iii) enucleating said at least one oocyte;

(iv) transferring the desired differentiated cell or cell nucleus into the enucleated oocyte;

(v) simultaneously fusing and activating the cell couplet to form a transgenic embryo in the presence of Adenophostin A and PLCζ

;

(vi) culturing said activated transgenic embryo until greater than the 2-cell developmental stage; and

(vii) transferring said transgenic embryo into a host mammal such that the embryo develops into a fetus.

98. The method of claim 97, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from mesoderm.

99. The method of claim 97, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from endoderm.

100. The method of claim 97, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from ectoderm.

101. The method of claim 97, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from fetal somatic tissue.

102. The method of claim 97, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from fetal somatic cells.

103. The method of claim 97, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from a fibroblast.

104. The method of claim 97, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from an ungulate.

105. The method of claim 97, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from an adult non-human mammalian somatic cell.

106. The method of claim 97, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is selected from the group consisting of epithelial cells, neural cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, B-lymphocytes, T-lymphocytes, erythrocytes, macrophages, monocytes, fibroblasts, and muscle cells.

107. The method of claim 97, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from an organ selected from the group consisting of skin, lung, pancreas, liver, stomach, intestine, heart, reproductive organ, bladder, kidney and urethra.

108. The method of claim 97, wherein said at least one oocyte is matured in vivo prior to enucleation.

109. The method of claim 97, wherein said at least one oocyte is matured in vitro prior to enucleation.

110. The method of claim 97, wherein said non-human mammal is a rodent.

111. The method of claim 97, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is a non-quiescent somatic cell or a nucleus isolated from said non-quiescent somatic cell.

112. The method of claim 97, wherein the fetus develops into an offspring.

113. The method of claim 97, wherein said at least one oocyte is enucleated about 10 to 60 hours after initiation of in vitro maturation.

114. The method of claim 97, wherein a desired gene is inserted, removed or modified in said differentiated mammalian cell or cell nucleus prior to insertion of said differentiated mammalian cell or cell nucleus into said enucleated oocyte.

115. The resultant offspring of the methods of claim 114.

116. The method of claim 97, wherein cytocholasin-B is used in the cloning protocol.

117. The method of claim 97, wherein cytocholasin-B is not used in the cloning protocol.

118. A method for cloning a non-human mammal through a nuclear transfer process comprising:

(i) obtaining desired differentiated mammalian cells to be used as a source of donor nuclei;

(ii) obtaining at least one oocyte from a mammal of the same species as the cells which are the source of donor nuclei;

(iii) enucleating said at least one oocyte;

(iv) transferring the desired differentiated cell or cell nucleus into the enucleated oocyte;

(v) simultaneously fusing and activating the cell couplet to form a transgenic embryo in the presence of PLCζ and

caprine sperm factor;

(vi) culturing said activated transgenic embryo until greater than the 2-cell developmental stage; and

(vii) transferring said transgenic embryo into a host mammal such that the embryo develops into a fetus.

119. The method of claim 118, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from mesoderm.

120. The method of claim 118, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from endoderm.

121. The method of claim 118, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from ectoderm.

122. The method of claim 118, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from fetal somatic tissue.

123. The method of claim 118, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from fetal somatic cells.

124. The method of claim 118, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from a fibroblast.

125. The method of claim 118, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from an ungulate.

126. The method of claim 118, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from an adult non-human mammalian somatic cell.

127. The method of claim 118, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is selected from the group consisting of epithelial cells, neural cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, B-lymphocytes, T-lymphocytes, erythrocytes, macrophages, monocytes, fibroblasts, and muscle cells.

128. The method of claim 118, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is from an organ selected from the group consisting of skin, lung, pancreas, liver, stomach, intestine, heart, reproductive organ, bladder, kidney and urethra.

129. The method of claim 118, wherein said at least one oocyte is matured in vivo prior to enucleation.

130. The method of claim 118, wherein said at least one oocyte is matured in vitro prior to enucleation.

131. The method of claim 118, wherein said non-human mammal is a rodent.

132. The method of claim 118, wherein said donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is a non-quiescent somatic cell or a nucleus isolated from said non-quiescent somatic cell.

133. The method of claim 118, wherein the fetus develops into an offspring.

134. The method of claim 118, wherein said at least one oocyte is enucleated about 10 to 60 hours after initiation of in vitro maturation.

135. The method of claim 118, wherein a desired gene is inserted, removed or modified in said differentiated mammalian cell or cell nucleus prior to insertion of said differentiated mammalian cell or cell nucleus into said enucleated oocyte.

136. The resultant offspring of the method of claim 135.

137. The method of claim 118, wherein cytocholasin-B is used in the cloning protocol.

138. The method of claim 118, wherein cytocholasin-B is not used in the cloning protocol.