电穿孔法转染哺乳动物细胞

nikos

站长注：下文是发表于Nature Methods中的一篇关于电穿孔转染方法的文章，站长对其作了注释，方便大家理解。电穿孔转染理论上可转染所有的组织细胞，因此对其他如脂质体、磷酸钙沉淀等方法转染效果不明显的细胞可选用此方法。电转过程中，最重要的就是电穿孔仪的电压、电容以及与电泳缓冲液的选择。提到电转仪，最出名的恐怕就属BIO-RAD了，他在1986年推出了世界上第一台电穿孔仪，并发布了多种细胞仪电转过程中的电压，电容，电转缓冲液等可省却大家很多的摸索过程，具体资料可到其主页上查找相关protocol(http://www3.bio-rad.com/B2B/vani ... hcfngcfkmdhkkdfll.0)。5 Z( M/ `( X) H. o2 ]( b! Q/ D; G7 [
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Nature Methods 3, 67 - 68 (2006) 9 {& E: y7 P  m5 d; I
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Transfection of mammalian cells by electroporation
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& `/ }7 H/ e* A+ C+ [Pulsed electrical fields can be used to introduce DNA into a wide variety of animal cells1, 2. Electroporation works well with cell lines that are refractive to other techniques, such as calcium phosphate–DNA coprecipitation. But as with other transfection methods, the optimal conditions for electroporation of untested cell lines must be determined experimentally. 0 E. c2 E9 [5 u2 k2 ]
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电穿孔转染可用于磷酸钙转染效率低下的细胞，具有操作简便，转染效率高等优点，但对于不同的细胞，其转染条件有很大的不同，需要进行摸索。6 \. M  A0 }: ?5 {, a* ~2 Y
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Procedure# L! A6 p- u' P6 j' E2 m
Preparation of the cells6 d+ ?" t3 ^4 f

6 `, g) x9 q3 o1.        Collect the cells to be transfected from cultures in the mid- to late-logarithmic phase of growth. Use either a rubber policeman or trypsin to release adherent cells. Centrifuge at 500g at 4 °C for 5 min.& A; E( o3 s1 s- w0 E1 D

1 |2 G, @, v. b( V, w在对数生长期收集细胞，离心沉淀。9 F8 [$ r: {/ A; h; Z) I% c
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2.        Resuspend the cell pellet in 0.5 volume of the original growth medium and measure the cell number using a hemocytometer.8 G8 m$ I6 v( b2 b5 P: w
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用培养基重悬细胞，计数
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% S" [& @: k: v% q' n3.        Collect the cells by centrifugation, as described in Step 1 and resuspend them in growth medium or phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 2 mM KH2PO4) at 15–25 °C at a concentration of 2.5 106 to 2.5 107 cells/ml.8 E3 e5 a0 T" y# f
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离心收集细胞，重悬细胞于培养基或PBS中，调整细胞密度至2.5 106 to 2.5 107 cells/ml.) ^! a; I5 ~. C6 P) z  ~# m; |
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4.        Transfer 400ul aliquots of the cell suspension (106–107 cells) into as many labeled electroporation cuvettes as needed. Place the loaded cuvettes on ice.
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8 E& S# a+ q. B0 c8 j  `7 P转移400ul细胞悬液至电转杯中，将之放在冰上。% Y# G5 d: |( o% h: H

7 x' t, C9 o/ J0 j7 Z# F7 v) s5.        Set the parameters on the electroporation device. (A typical capacitance value is 1,050 F.) Voltages range from 200 to 350 V, depending on the cell line, but generally average 260 V. Use an infinite internal resistance value. Discharge a blank cuvette containing PBS at least twice before electroporating cells.
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设置电转参数，一般电容为1050 F，电压范围200到 350 V（因细胞不同而不同），一般260V。在电穿孔之前，需用含PBS的空电转杯通电两次。
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Introduction of the DNA2 V* D8 h( m) \' f1 h

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6.        Add 10–30 g of plasmid DNA in a volume of up to 40 l to each cuvette containing cells. (Some investigators add carrier DNA (for example, salmon sperm DNA) to bring the total amount of DNA to 120 g.) Gently mix the cells and DNA by pipetting the solution up and down. Proceed to Step 7 without delay.* Q% S* @" C2 u5 p, {; Z# ^5 C+ g
Do not introduce air bubbles into the suspension during the mixing step.
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+ Q3 ^/ I5 C1 |; m. n% g- k加入10–30 g质量DNA于含有细胞的电转杯中，不超过40ul，轻轻混匀后，立即进入下一步
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7.         Immediately transfer the cuvette to the electroporator and discharge the device. After 1–2 min, remove the cuvette, place it on ice and proceed immediately to the next step.
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& ^! P+ m! v/ y/ I2 c将电转杯放入电转仪中，通电。1-2分钟之后，取出放在冰上，立即进入下一步操作。
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' f9 P$ t$ }; ]8.        Transfer the electroporated cells to a 35-mm culture dish using a micropipettor equipped with a sterile tip. Rinse out the cuvette with a fresh aliquot of growth medium and add the washings to the culture dish. Transfer the dish to a humidified incubator at 37 °C with an atmosphere of 5–7% CO2.+ U( D/ O* k; ~4 Z- R$ _1 p

# w* i' v: P/ c9 ^7 I$ F7 J4 v将细胞转入培养皿中放入培养箱培养。/ c% u5 ~# Z( a, U* I6 X% j

& D9 G/ d6 q4 d' Z1 S8 p" O9.        Repeat Steps 6–8 until all of the DNA cell samples have been treated. Recording the actual pulse time for each cuvette will facilitate comparisons between experiments.
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. b0 d* @! S5 q7 x4 f7 d2 x6 l: i重复6–8步，将各组细胞电转。
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* k( j5 |4 ?7 R* R2 F10.    If the objective is stable transformation of the cells, proceed directly to Step 11. For transient expression, examine the cells 24–96 h after electroporation using an appropriate assay.
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& k9 X3 p, M8 I如果是要获得稳定转染细胞，直接进行第十一步。暂时转染，在电穿孔后24–96 h后检测。
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11.    To isolate stable transfectants, incubate for 48–72 h in complete medium, trypsinize the cells and replate them in the appropriate selective medium. Change the selective medium every 2–4 d for 2–3 weeks to remove the debris of dead cells and to allow colonies of resistant cells to grow. Thereafter, clone individual colonies and propagate for the appropriate assay.
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要分离稳定转染的细胞，在完全培养基中培养48–72 h，胰酶消化后用选择培养基培养。, t8 M* C0 y2 q  ?) j* B  `" h
Source
% p, g4 q8 B7 @3 n; u6 ZThis protocol was adapted from "DNA transfection by electroporation" in Molecular Cloning: A Laboratory Manual (eds. Sambrook, J. & Russell, D.W.) 16.33–16.36 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001; http://www.cshlpress.com/link/molclon3.htm).
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! {* M2 i# Q( tREFERENCES . q5 Z2 |2 H- X9 c0 X- u

6 C6 P( J7 V* y- S. A- P, x1.      Wong, T.-K. & Neumann, E. Electric field mediated gene transfer. Biochem. Biophys. Res. Commun. 107, 584–587 (1982). | PubMed | ISI | ChemPort |
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2.      Potter, H. , Weir, L. & Leder, P. Enhancer-dependent expression of human immunoglobulin genes introduced into mouse pre-B lymphocytes by electroporation. Proc. Natl. Acad. Sci. USA 81, 7161–7165 (1984). | PubMed | ChemPort |

zpzp0312

学习了！~~~

POPOLD

有难度  不知转染效率如何

cl5823283

好啊，正想学习。转染效率是多少啊，我也想知道！

张也行

是啊，电转的效率不是挺低的吗？

wxl87

好贴，以后能用到啊

懵懂干细胞

楼主能否把NATURE METHODS 的文献上传一下啊！! O% O0 M6 K8 r' |+ g. S: X

siyue13

不是吧，电转的效率目前应该是所有方法里面最高的吧，怎么说一台电转仪也要小十万呢

gh2010

我第一次转干细胞的，大概才1/1000，是不是很低啊？
2 g+ P' `. E: Q% k! R& [1 p7 B还有，我电转后直接把细胞稀释到几个孔里培养的，这样会不会影响转染效率？

echowdk

回复 gh2010 的帖子
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1/1000那是有够低的，不管你是做过表达还是做敲除。
4 W4 W0 d. x  e: V7 p$ c6 U! V干细胞电转一次需要的细胞量是0.75*107个，电转过程会有60%左右的细胞死亡，但是电之后铺到10cm的培养皿上培养完全没有问题。! b/ l- b: C& ?, w+ F/ c
另外，整个电转过程的时间也是要把握好的，一般不能超过30min。