Electroporation of ES Cell 胚胎干细胞电穿孔[Mount Sinai Hospital]

肖爱华

Electroporation of ES Cell Nagy Lab,Samuel Lunenfeld Research Institute, Room 881 ,Mount Sinai Hospital
9 {! f% Q2 M' I) x0 r0 G) R, }  U胚胎干细胞电穿孔，该实验室专著于小鼠遗传学以及其跟人类疾病关系
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Electroporation of ES cells
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, ^4 @  D, O1 D+ Q4 p2 R# JCells are routinely passaged two days prior to electroporating. Usually one 10 cm plate at approximately 80% confluency will provide enough cells for 1-2 electroporations.
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Procedure* K4 }3 E' u5 H4 U# ^8 z
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1. Change medium on ES cells 3-4 hours prior to electroporation
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2. Gelatinize 10 cm plates, then add 10 ml medium to each. + I0 T3 W" x! Z1 |" L4 f1 ]
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3. Place them in a 37 0C incubator until they are required.
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4. Switch on the electroporation apparatus. , o: D  ~- h- C, j% @$ D, |' d

% |* u' A# `" e' z3 n5. Harvest ES cells by trypsinization.
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% a' T* J& J- W% K& ?3 }6. Resuspend the cell pellet in ice-cold PBS (1 ml for each 10 cm plate). / \2 b0 R- E+ H4 E& U( j
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7. Determine the cell density (haemocytometer) and dilute with PBS to the required density for electroporation. We regularly electroporate at a relatively high cell density: 7x106 cells/ml (this number varies between different labs). + W4 n+ [: K0 u2 q3 i
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8. For each electroporation mix together 20-40 microgram (1 礸/祃) DNA (for an approximately 10 kb vector) and 0.8 ml of the ES cell suspension in an electroporation cuvette (BioRad, Cat. No. 165-2088). 3 ?# c. J; N: K, A! ^1 y% O. Y
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9. Set up the electroporation conditions prior to placing the cuvette into the electroporation chamber. We routinely use 250 V, 500 microF for the BioRad GenePulser.
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8 M. `) k0 y( w8 B8 f  [/ d10. Zap the cuvette, then place it on ice for 20 min to 1 hour. ; t5 q# O( b" p3 v$ \! g% H4 O
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11. Transfer the cells from the cuvette into the prewarmed medium containing dishes. (The contents of one cuvette are routinely seeded into two 10 cm dishes).
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2 k  m+ x) ?3 g- K/ A9 f+ x' O$ `9 Cpost electroporation:5 ?2 {" Y% i) q0 Z- B0 k5 v
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12. Change medium daily.
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13. If drug selection is required start this on the second day after electroporation. 8 }. E1 F9 P# v" f$ x

, ?+ o, c& g7 s  `; F# s' m14. Continue the selection until colonies become apparent, and grow to a size that is amenable to picking (usually takes 7-10 days).

wuhonglian2008

挺好的谢谢了！