Reverse transfection method

• US 6,544,790 B1
• Filed: 09/18/2000
• Issued: 04/08/2003
• Est. Priority Date: 09/17/1999
• Status: Expired due to Term

• Current Assignee: Whitehead Institute for Biomedical Research
• Original Assignee: Whitehead Institute for Biomedical Research
• Inventors: Sabatini, David M.
• Primary Examiner(s): Yucel, Remy
• Assistant Examiner(s): KATCHEVES, KONSTANTINA T
• Application Number:
  US09/664,297
• Time in Patent Office: 932 Days
• Field of Search: 435/6, 435/440, 435/320.1, 435/7.21, 435/455, 435/174, 422/50
• US Class Current: 435/455
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• CPC Class Codes: C12N 15/88 using microencapsulation, e.g. using amphiphile liposome vesicle
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

A reverse transfection method of introducing DNA of interest into cells and arrays, including microarrays, of reverse transfected cells.

Claims

1. A method of introducing nucleic acid molecules into mammalian cells comprising:

(a) depositing a plurality of nucleic acid molecule-containing mixtures onto a surface in discrete, defined locations, wherein each of the nucleic acid molecule-containing mixtures comprises a nucleic acid molecule to be introduced into mammalian cells and a gelatin;

(b) allowing the nucleic acid molecule-containing mixtures to dry on the surface, thereby producing a surface having the nucleic acid molecule-containing mixtures affixed thereon in discrete, defined locations; and

(c) plating the mammalian cells onto the surface at a density of 0.3×

10⁵/cm² to 3.0×

10⁵/cm² under appropriate conditions for entry of the nucleic acid molecules in the nucleic acid molecule-containing mixtures into mammalian cells;

whereby the nucleic acid molecules are introduced into the mammalian cells in the location in which each of the nucleic acid molecule-containing mixtures was deposited.

2. The method of claim 1, further comprising the steps after step (b) of:

(i) covering the surface bearing the nucleic acid molecule-containing mixtures with an appropriate amount of a lipid-based transfection reagent and maintaining the resulting product under conditions appropriate for complex formation between the nucleic acid molecules in the nucleic acid molecule-containing mixture and the transfection reagent; and

(ii) removing the non-complexed transfection reagent.

3. The method of claim 1, wherein said nucleic acid molecule-containing mixtures further comprise a sugar, a buffer that facilitates nucleic acid molecule condensation, and an appropriate lipid-based transfection reagent.

4. The method of any one of claims 1-3, wherein each nucleic acid molecule to be introduced is contained in a vector.

5. The method of claim 4, wherein the vector is an episomal vector or a chromosomally integrated vector.

6. The method of claim 4, wherein the vector is a plasmid or a viral-based vector.

7. The method of claim 6, wherein the vector is an expression vector.

8. The method of claim 7, wherein said nucleic acid molecules are expressed in the mammalian cells.

9. The method of any one of claims 1-3, wherein the surface is glass, polystyrene or plastic, optionally coated with poly-L-lysine.

10. The method of any one of claims 1-3, wherein the surface is the surface of a slide.

11. The method of claim 10, wherein the slide is a glass slide coated with poly-L-lysine or a gamma-amino propyl silane slide.

12. The method of any one of claims 1-3, wherein the cells are plated at a density of 0.5×

10⁵/cm² to 2.0×

10⁵/cm².

13. The method of claim 12, wherein the cells are plated at a density of 0.5×

10⁵/cm² to 1.0×

10⁵/cm².

14. The method of any one of claims 1-3, wherein said nucleic acid molecule is an oligonucleotide, DNA, or RNA.

15. The method of claim 14, wherein said nucleic acid molecule is DNA.

16. The method of claim 1 or 2, wherein said nucleic acid molecule is DNA and wherein the concentration of said DNA is 0.01 μ

g/μ

l to 0.5 μ

g/μ

l.

17. The method of claim 15, wherein the concentration of said DNA is 0.02 μ

g/μ

l to 0.1 μ

g/μ

l.

18. The method of claim 3, wherein said nucleic acid molecule is DNA and wherein the concentration of said DNA is 0.1 μ

g/μ

l to 2.0 μ

/μ

l.

19. The method of any one of claims 1-3, wherein said nucleic acid molecules encode polypeptides that are expressed in the mammalian cells.

20. The method of any one of claims 1-3, wherein said nucleic acid molecules, when introduced into the mammalian cells, inhibit a function of a gene in the mammalian cells.

21. The method of claim 1, wherein the gelatin is selected from the group consisting of a protein gelatin, a hydrogel, a sugar-based gelatin or a synthetic gelatin.

22. The method of claim 21, wherein the gelatin is present at a concentration in the nucleic acid molecule-containing mixture ranging from about 0.05% to about 0.5%.

23. The method of claim 22, wherein the concentration of gelatin is from about 0.1% to about 0.2%.

24. The method of claim 2, wherein the gelatin is selected from the group consisting of a protein gelatin, a hydrogel, a sugar-based gelatin or a synthetic gelatin.

25. The method of claim 24, wherein the gelatin is present at a concentration in the nucleic acid molecule-containing mixture ranging from about 0.05% to about 0.5%.

26. The method of claim 25, wherein the concentration of gelatin is from about 0.1% to about 0.2%.

27. The method of claim 3, wherein the gelatin is selected from the group consisting of a protein gelatin, a hydrogel, a sugar-based gelatin or a synthetic gelatin.

28. The method of claim 22, wherein the gelatin is present at a concentration in the nucleic acid molecule-containing mixture ranging from about 0.01% to 0.05%.

29. The method of claim 23, wherein said sugar is sucrose ranging in concentration from about 0.1M to about 0.4M.

30. The method of any one of claims 21, 24, or 27, wherein the gelatin is Type B gelatin.

31. The method of any one of claims 21, 24, or 27, wherein each nucleic acid molecule to be introduced is contained in a vector, and the surface is a slide.

32. The method of claim 31, wherein the vector is an expression vector and said nucleic acid molecules are expressed in the mammalian cells.

33. The method of any one of claims 1-3, wherein at least two different nucleic acid molecules are co-transfected into a mammalian cell.

34. The method of any one of claims 1-3, wherein said affixed plurality of nucleic acid molecules form an array of nucleic acid molecules and wherein said cells into which the nucleic acid molecules are introduced form an array of cells comprising the nucleic acid molecules.

35. The method of claim 34, wherein the gelatin is selected from the group consisting of a protein gelatin, a hydrogel, a sugar-based gelatin or a synthetic gelatin.

36. The method of claim 34, wherein the array comprises at least 96 different discrete, defined locations of known sequence composition.

37. The method of claim 36, wherein the array comprises at least 192 different discrete, defined locations of known sequence composition.

38. The method of claim 37, wherein the array comprises up to 10,000 to 15,000 different discrete, defined locations of known sequence composition.

39. The method of claim 34, wherein each of the defined locations is 100-200 μ

m in diameter.

40. The method of claim 39, wherein each of the defined locations is 200-500 μ

m apart from each other.

148. An array produced by the method of claim 34.

41. A method of introducing DNA molecules into eukaryotic cells comprising:

(a) depositing a plurality of DNA molecule-containing mixtures onto a surface in discrete, defined locations, wherein each of the DNA molecule-containing mixtures comprises a DNA molecule to be introduced into eukaryotic cells and a gelatin, wherein the concentration of DNA in said DNA molecule-containing mixtures is 0.01 μ

g/μ

L to 0.5 μ

g/μ

L;

(b) allowing the DNA molecule-containing mixtures to dry on the surface, thereby producing a surface having the DNA molecule-containing mixtures affixed thereon in discrete, defined locations; and

(c) plating the eukaryotic cells onto the surface in sufficient density and under appropriate conditions for entry of the DNA molecules in the DNA molecule-containing mixtures into eukaryotic cells;

whereby the DNA molecules are introduced into the eukaryotic cells in the location in which each of the nucleic acid molecule-containing mixtures was deposited.

42. The method of claim 41, further comprising the steps after step (b) of:

(i) covering the surface bearing the DNA molecule-containing mixtures with an appropriate amount of a lipid-based transfection reagent and maintaining the resulting product under conditions appropriate for complex formation between the DNA molecules in the DNA molecule-containing mixture and the transfection reagent; and

(ii) removing the non-complexed transfection reagent.

43. The method of claim 41, wherein said DNA molecule-containing mixtures further comprise a sugar, a buffer that facilitates DNA molecule condensation, and an appropriate lipid-based transfection reagent.

44. The method of any one of claims 41-43, wherein each DNA molecule to be introduced is contained in a vector.

45. The method of claim 44, wherein the vector is an episomal vector or a chromosomally integrated vector.

46. The method of claim 44, wherein the vector is a plasmid or a viral-based vector.

47. The method of claim 46, wherein the vector is an expression vector.

48. The method of claim 47, wherein said DNA molecules are expressed in the eukaryotic cells.

49. The method of any one of claims 41-43, wherein the surface is glass, polystyrene or plastic, optionally coated with poly-L-lysine.

50. The method of any one of claims 41-43, wherein the surface is the surface of a slide.

51. The method of claim 50, wherein the slide is a glass slide coated with poly-L-lysine or a gamma-amino propyl silane slide.

52. The method of any one of claims 41-43, wherein the eukaryotic cells are mammalian cells.

53. The method of any one of claims 41-43, wherein said DNA molecules encode polypeptides that are expressed in the eukaryotic cells.

54. The method of any one of claims 41-43, wherein said DNA molecules, when introduced into the eukaryotic cells, inhibit a function of a gene in the eukaryotic cell.

55. The method of claim 41, wherein the gelatin is selected from the group consisting of a protein gelatin, a hydrogel, a sugar-based gelatin or a synthetic gelatin.

56. The method of claim 55, wherein the gelatin is present at a concentration in the DNA molecule-containing mixture ranging from about 0.05% to about 0.5%.

57. The method of claim 56, wherein the concentration of gelatin is from about 0.1% to about 0.2%.

58. The method of claim 42, wherein the gelatin is selected from the group consisting of a protein gelatin, a hydrogel, a sugar-based gelatin or a synthetic gelatin.

59. The method of claim 58, wherein the gelatin is present at a concentration in the DNA molecule-containing mixture ranging from about 0.05% to about 0.5%.

60. The method of claim 58, wherein the concentration of gelatin is from about 0.1% to about 0.2%.

61. The method of claim 43, wherein the gelatin is selected from the group consisting of a protein gelatin, a hydrogel, a sugar-based gelatin or a synthetic gelatin.

62. The method of claim 61, wherein the gelatin is present at a concentration in the DNA molecule-containing mixture is from about 0.01% to 0.05%.

63. The method of claim 62, wherein said sugar is sucrose ranging in concentration from about 0.1M to about 0.4M.

64. The method of any one of claims 55, 56, or 61, wherein the gelatin is Type B gelatin.

65. The method of any one of claims 55, 56 or 61, wherein the eukaryotic cells are plated at high density onto the surface, each DNA molecule to be introduced is contained in a vector, and the surface is a slide.

66. The method of claim 65, wherein the vector is an expression vector and said DNA molecules are expressed in the eukaryotic cells.

67. The method of any one of claims 41-43, wherein at least two different DNA molecules are co-transfected into a eukaryotic cell.

68. The method of any one of claims 41-43, wherein said affixed plurality of DNA molecules form an array of DNA molecules and wherein said cells into which the DNA molecules are introduced form an array of cells comprising the DNA molecules.

69. The method of claim 68, wherein the gelatin is selected from the group consisting of a protein gelatin, a hydrogel, a sugar-based gelatin or a synthetic gelatin.

70. The method of claim 68, wherein the array comprises at least 96 different discrete, defined locations of known sequence composition.

71. The method of claim 70, wherein the array comprises at least 192 different discrete, defined locations of known sequence composition.

72. The method of claim 71, wherein the array comprises up to 10,000 to 15,000 different discrete, defined locations of known sequence composition.

73. The method of claim 68, wherein each of the defined locations is 100-200 μ

m in diameter.

74. The method of claim 73, wherein each of the defined locations is 200-500 μ

m apart from each other.

75. The method of claim 68, wherein said DNA molecules encode polypeptides that are expressed in the eukaryotic cells.

76. The method of claim 68, wherein said DNA molecules, when introduced into the eukaryotic cells, inhibit a function of a gene in the eukaryotic cells.

149. An array produced by the method of claim 68.

77. A method of introducing nucleic acid molecules into eukaryotic cells comprising:

(a) depositing a plurality of nucleic acid molecule-containing mixtures onto a surface in discrete, defined locations, wherein each of the nucleic acid molecule-containing mixtures comprises a nucleic acid molecule to be introduced into eukaryotic cells and a gelatin, wherein the gelatin is selected from the group consisting of a protein gelatin, a hydrogel, a sugar-based gelatin or a synthetic gelatin, and is present at a concentration from about 0.05% to about 0.5%;

(b) allowing the nucleic acid molecule-containing mixtures to dry on the surface, thereby producing a surface having the nucleic acid molecule-containing mixtures affixed thereon in discrete, defined locations; and

(c) plating the eukaryotic cells onto the surface in sufficient density and under appropriate conditions for entry of the nucleic acid molecules in the nucleic acid molecule-containing mixtures into eukaryotic cells;

whereby the nucleic acid molecules are introduced into the eukaryotic cells in the location in which each of the nucleic acid molecule-containing mixtures was deposited.

78. The method of claim 77, further comprising the steps after step (b) of:

(i) covering the surface bearing the nucleic acid molecule-containing mixtures with an appropriate amount of a lipid-based transfection reagent and maintaining the resulting product under conditions appropriate for complex formation between the nucleic acid molecules in the nucleic acid molecule-containing mixture and the transfection reagent; and

(ii) removing the non-complexed transfection reagent.

79. The method of claim 77, wherein said nucleic acid molecule-containing mixtures further comprise a sugar, a buffer that facilitates nucleic acid molecule condensation, and an appropriate lipid-based transfection reagent.

80. The method of any one of claims 77-79, wherein each nucleic acid molecule to be introduced is contained in a vector.

81. The method of claim 80, wherein the vector is an episomal vector or a chromosomally integrated vector.

82. The method of claim 80, wherein the vector is a plasmid or a viral-based vector.

83. The method of claim 82, wherein the vector is an expression vector.

84. The method of claim 83, wherein said nucleic acid molecules are expressed in the eukaryotic cells.

85. The method of any one of claims 78-79, wherein the surface is glass, polystyrene or plastic, optionally coated with poly-L-lysine.

86. The method of any one of claims 77-78, wherein the surface is the surface of a slide.

87. The method of claim 86, wherein the slide is a glass slide coated with poly-L-lysine or a gamma-amino propyl silane slide.

88. The method of any one of claims 77-79, wherein the eukaryotic cells are mammalian cells.

89. The method of any one of claims 77-79, wherein said nucleic acid molecule is an oligonucleotide, DNA, or RNA.

90. The method of claim 89, wherein said nucleic acid molecule is DNA.

91. The method of any one of claims 77-79, wherein said nucleic acid molecules encode polypeptides that are expressed in the eukaryotic cells.

92. The method of any one of claims 77-79, wherein said nucleic acid molecules, when introduced into the eukaryotic cells, inhibit a function of a gene in the eukaryotic cell.

93. The method of claim 77, wherein the concentration of gelatin is from about 0.1% to about 0.2%.

94. The method of claim 78, wherein the gelatin is selected from the group consisting of a protein gelatin, a hydrogel, a sugar-based gelatin or a synthetic gelatin.

95. The method of claim 94, wherein the concentration of gelatin is from about 0.1% to about 0.2%.

96. The method of claim 79, wherein the gelatin is selected from the group consisting of a protein gelatin, a hydrogel, a sugar-based gelatin or a synthetic gelatin.

97. The method of claim 96, wherein said sugar is sucrose ranging in concentration from about 0.1M to about 0.4M.

98. The method of any one of claims 77, 94, or 96, wherein the gelatin is Type B gelatin.

99. The method of any one of claims 77, 94, or 96, wherein the eukaryotic cells are plated at high density onto the surface, each nucleic acid molecule to be introduced is contained in a vector, and the surface is a slide.

100. The method of claim 99, wherein the vector is an expression vector and said nucleic acid molecules are expressed in the eukaryotic cells.

101. The method of any one of claims 77-79, wherein at least two different nucleic acid molecules are co-transfected into a eukaryotic cell.

102. The method of any one of claims 77-79, wherein said affixed plurality of nucleic acid molecules form an array of nucleic acid molecules and wherein said cells into which the nucleic acid molecules are introduced form an array of cells comprising the nucleic acid molecules.

103. The method of claim 102, wherein the gelatin is selected from the group consisting of a protein gelatin, a hydrogel, a sugar-based gelatin or a synthetic gelatin.

104. The method of claim 102, wherein the array comprises at least 96 different discrete, defined locations of known sequence composition.

105. The method of claim 104, wherein the array comprises at least 192 different discrete, defined locations of known sequence composition.

106. The method of claim 105, wherein the array comprises up to 10,000 to 15,000 different discrete, defined locations of known sequence composition.

107. The method of claim 103, wherein each of the defined locations is 100-200 μ

m in diameter.

108. The method of claim 107, wherein each of the defined locations is 200-500 μ

m apart from each other.

109. The method of claim 107, wherein said nucleic acid molecules are DNA or RNA.

110. The method of claim 107, wherein said nucleic acid molecules encode polypeptides that are expressed in the eukaryotic cells.

111. The method of claim 107, wherein said nucleic acid molecules, when introduced into the eukaryotic cells, inhibit a function of a gene in the eukaryotic cell.

150. An array produced by the method of claim 102.

112. A method of introducing nucleic acid molecules into eukaryotic cells comprising:

(a) depositing a plurality of nucleic acid molecule-containing mixtures onto a surface in discrete, defined locations, wherein each of the nucleic acid molecule-containing mixtures comprises;

(i) a nucleic acid molecule to be introduced into eukaryotic cells,

(ii) a gelatin, wherein the gelatin is selected from the group consisting of a protein gelatin, a hydrogel, a sugar-based gelatin or a synthetic gelatin, and is present at a concentration from about 0.01% to about 0.05%,

(iii) a sugar,

(iv) a buffer that facilitates nucleic acid molecule condensation, and

(v) a lipid-based transfection reagent;

(b) allowing the nucleic acid molecule-containing mixtures to dry on the surface, thereby producing a surface having the nucleic acid molecule-containing mixtures affixed thereon in discrete, defined locations; and

(c) plating the eukaryotic cells onto the surface in sufficient density and under appropriate conditions for entry of the nucleic acid molecules in the nucleic acid molecule-containing mixtures into eukaryotic cells;

whereby the nucleic acid molecules are introduced into the eukaryotic cells in the location in which each of the nucleic acid molecule-containing mixtures was deposited.

113. The method of claim 112, further comprising the steps after step (b) of:

(i) covering the surface bearing the nucleic acid molecule-containing mixtures with an appropriate amount of a lipid-based transfection reagent and maintaining the resulting product under conditions appropriate for complex formation between the nucleic acid molecules in the nucleic acid molecule-containing mixture and the transfection reagent; and

(ii) removing the non-complexed transfection reagent.

114. The method of claim 112 or 228, wherein each nucleic acid molecule to be introduced is contained in a vector.

115. The method of claim 114, wherein the vector is an episomal vector or a chromosomally integrated vector.

116. The method of claim 114, wherein the vector is a plasmid or a viral-based vector.

117. The method of claim 116, wherein the vector is an expression vector.

118. The method of claim 117, wherein said nucleic acid molecules are expressed in the eukaryotic cells.

119. The method of claim 112 or 113, wherein the surface is glass, polystyrene or plastic, optionally coated with poly-L-lysine.

120. The method of claim 112 or 113, wherein the surface is the surface of a slide.

121. The method of claim 120, wherein the slide is a glass slide coated with poly-L-lysine or a gamma-amino propyl silane slide.

122. The method of claim 112 or 113, wherein the eukaryotic cells are mammalian cells.

123. The method of claim 122, wherein the cells are plated at a density of 0.3×

10⁵/cm² to 3.0×

10⁵/cm².

124. The method of claim 123, wherein the cells are plated at a density of 0.5×

10⁵/cm² to 2.0×

10⁵/cm².

125. The method of claim 124, wherein the cells are plated at a density of 0.5×

10⁵/cm² to 1.0×

10⁵/cm².

126. The method of claim 112 or 113, wherein said nucleic acid molecule is an oligonucleotide, DNA, or RNA.

127. The method of claim 126, wherein said nucleic acid molecule is DNA.

128. The method of claim 112 or 113, wherein said nucleic acid molecule is DNA and wherein the concentration of said DNA is 0.01 μ

g/μ

l to 0.5 μ

g/μ

l.

129. The method of claim 128, wherein the concentration of said DNA is 0.02 μ

g/μ

l to 0.1 μ

g/μ

l.

130. The method of claim 112 or 113, wherein said nucleic acid molecules encode polypeptides that are expressed in the eukaryotic cells.

131. The method of claim 112 or 113, wherein said nucleic acid molecules, when introduced into the eukaryotic cells, inhibit a function of a gene in the eukaryotic cell.

132. The method of claim 113, wherein the gelatin is selected from the group consisting of a protein gelatin, a hydrogel, a sugar-based gelatin or a synthetic gelatin.

133. The method of claim 112 or 113, wherein the gelatin is Type B gelatin.

134. The method of claim 132, wherein the eukaryotic cells are plated at high density onto the surface, each nucleic acid molecule to be introduced is contained in a vector, and the surface is a slide.

135. The method of claim 134, wherein the vector is an expression vector and said nucleic acid molecules are expressed in the eukaryotic cells.

136. The method of claim 112 or 113, wherein said sugar is sucrose ranging in concentration from about 0.1M to about 0.4M.

137. The method of claim 112 or 113, wherein at least two different nucleic acid molecules are co-transfected into a eukaryotic cell.

138. The method of claim 112 or 113, wherein said affixed plurality of nucleic acid molecules form an array of nucleic acid molecules and wherein said cells into which the nucleic acid molecules are introduced form an array of cells comprising the nucleic acid molecules.

139. The method of claim 138, wherein the gelatin is selected from the group consisting of a protein gelatin, a hydrogel, a sugar-based gelatin or a synthetic gelatin.

140. The method of claim 138, wherein the array comprises at least 96 different discrete, defined locations of known sequence composition.

141. The method of claim 140, wherein the array comprises at least 192 different discrete, defined locations of known sequence composition.

142. The method of claim 141, wherein the array comprises up to 10,000 to 15,000 different discrete, defined locations of known sequence composition.

143. The method of claim 142, wherein each of the defined locations is 100-200 μ

m in diameter.

144. The method of claim 143, wherein each of the defined locations is 200-500 μ

m apart from each other.

145. The method of claim 138, wherein said nucleic acid molecules are DNA or RNA.

146. The method of claim 138, wherein said nucleic acid molecules encode polypeptides that are expressed in the eukaryotic cells.

147. The method of claim 138, wherein said nucleic acid molecules, when introduced into the eukaryotic cells, inhibit a function of a gene in the eukaryotic cell.

151. An array produced by the method of claim 138.