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293T and Pheonix cells grow in DMEM + 10%FBS. If you are transfecting other cells, you can use whatever medium those cells normally grow in and change to DMEM + 10%FBS on the day of the transfection. You can change back to “normal” medium 24 hours post-transfection. Calcium Phosphate transfection of M2182 in RPMI (w/o FBS) was reported to cause cells to die. All the transfection have been done in DMEM + 10% FBS, no transfection in other media was performed so far.

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[Bio101] Lentivirus and Retrovirus Transfection
[Bio101] 慢病毒和逆转录病毒转染

分子生物学 > DNA > 转染
作者: Yanlin Huang
3/5/2011, 19043 views, 3 Q&A
DOI: https://doi.org/10.21769/BioProtoc.38

[Abstract] 293T and Pheonix cells grow in DMEM + 10%FBS. If you are transfecting other cells, you can use whatever medium those cells normally grow in and change to DMEM + 10%FBS on the day of the transfection. You can change back to “normal” medium 24 hours post-transfection. Calcium Phosphate transfection of M2182 in RPMI (w/o FBS) was reported to cause cells to die. All the transfection have been done in DMEM + 10% FBS, no transfection in other media was performed so far.

[Abstract] 293T 和Pheonix细胞生长在含10%胎牛血清的DMEM中。如果你转染其他细胞,可以使用这些细胞平时使用的培养基,在转染的当天换成含10%胎牛血清的DMEM 。可以在转染24h后换回平时使用的培养基。有报道说,磷酸钙转染法转染M2182(使用无血清 RPMI 培养基)会导致细胞死亡。所有的转染都在含10%胎牛血清的DMEM中进行,迄今为止,未曾使用其他培养基进行转染。

Materials and Reagents

 

1.        Tissue culture plates with appropriate size (Fiosher).

2.        293T and Pheonix cell line

3.        Dulbecco's Modified Eagle Medium (D-MEM) (1X), liquid (high glucose) (Invitrogen)

4.        Fetal bovine serum (Hyclone)

5.        Packaging vector PcmvR8.74

6.        Envelop vector PMD2VSVG

7.        Reporter vector DsRED, GFP or LacZ

8.        2x HBS

9.        2M CaCl2 (Mallinkrodt Cat # 4160)

 

Equipments

 

1.        Tissue Culture Incubator

 

Procedure

 

1.        Day 0:

Plate 4.5x106 293T (or Pheonix for retrovirus) on 10cm plate. Incubate overnight at 37°C. You can also scale down to 6cm or 6 well plates.

Note: alternatively, you can plate the cells the same day as the transfection. See Day 1.

2.        Day 1: TRANSFECTION

Note: if you plated cells on the previous day, change medium to DMEM + 10% FBS 2hrs prior to transfection and skip to step 4.

1)     Plate 293T (or Pheonix for retrovirus) at ~50% confluence on 10cm TC plates. I calculate it roughly from the original plate confluence. Ex. I will plate 2 X 10cm plates from 1 100% confluence 10cm plate. I have scaled down to 6cm and 6 well plates. Just keep final confluence of cells at roughly 50%. 

2)     Incubate plates in 37°C incubator for ~3 to 5hrs to allow cells to attach.

3)     Thaw out all reagents to room temp before proceeding with transfection.

4)     Once cells attached, prepare DNA mix for transfection. For each 10cm plate, add the following to 5ml polypropylene tube:

a)       10μg DNA of interested (for retrovirus, 10μg DNA)

b)      6.5μg packaging vector (packaging vector already in Pheonix, so no need to add)

c)       3.5μg envelope vector (for retrovirus, 5μg envelope vector)

d)      0.1 to 0.2μg of reporter vector (optional) – GFP, DsRED, or LacZ

e)       Bring mixture up to 437.5μl with 0.1% TE in H2O

If you use different size plates, just scale down. See below table.

Note: You can make a master mix if you have multiple plates of the same transfection. However you need to do each transfection separately for high efficiency.

Note: the ratio of DNA:packaging vector: envelope vector is crucial for max viral titer. The ratio given was figured out by Weissman’s Lab. Please keep to this ratio as much as you can.

5)     Vortex the DNA mix on highest speed setting and add 62.5μl of 2M CaCl2. While still vortexing, add 500μl 2X HBS drop wise to the DNA/CaCl2 mix (roughly 2 drops/sec using p1000 pipettor).

6)     Immediately add HBS/DNA solution onto cells. Do this in a gentle, drop wise manner and spread it across cells in medium.

7)     In a few minutes you should be able to observe, under microscope, evenly distributed small black particles on top of the cells.

8)     Incubate cells in 37°C incubator overnight.

 

 

 2xHBS

2M CaCl2

DNA mix

DNA:Pack:Env

10cm plates

500μl

62.5μl

437.5μl

10μg:6.5μg:3.5μg

6cm plates

250μl

31μl

219μl

5μg:3.3μg:1.8μg

6 wells plate

83μl

10.5μl

73μl

1.5μg:1μg:0.6μg

 

3.        Day 2: 24 hours post-transfection

1)      Change medium to 10ml fresh DMEM 10% FBS. You can generally see the efficiency of your transfection by now if you used a reporter vector. At 0.2μg ofDsRED, I can usually observe >75% transfection efficiency. If you cant see much fluorescent cells at this time, wait until 48hrs post-transfection and observe again.

2)      Note: we have always package our viruses in 37°C incubator although it has been reported that virus is more stable if incubation is carried out at 32°C.

3)      If you want to titer the virus, plate target cells now for infection tomorrow. I often plate 3X10cm plates per virus for 3 different titrations.

4.        Day 3: 48 hours post-transfection

1)     Aliquot supernatant of transfected cells into desired volume and freeze at -80°C to kill any cells in the supernatant. Alternatively, you can filter the supethrough 0.45μm filter to remove the cells. You can also chose to centrifuge the supernatant first if there are lots of floating cells. However, you need to either freeze or filter the supe before using the virus to completely remove any chance of cell contamination.

2)     Store viruses at -80°C. 

If you are titering or infecting target cells, follow the rest of the protocol:

3)     Add 10μl 1000X polybrene (1000x = 5-8μg/ml) to each 10cm plate of target cells (10ml medium). Add 0.1μl, 1μl, or 10μl of virus supe to each plate of cells for titering your virus. Incubate at 37°C overnight.

5.        Day 4 : REMOVE VIRUS SUPERNATANT

1)     24 hours post-infection, change to fresh medium. Incubate overnight at 37°C.

6.        Day 5: 24-48 HOURS POST-INFECTION

1)     Cells are now ready to be assay for biochemical event of interest (ex. start selection with appropriate antibiotic). The actual reverse transcription and integration take place within 24-36 hours, depending on cell growth kinetics.

2)     Culture cells as normal.

 

Recipe

 

1.        2xHBS for calcium phosphate coprecipitation transfection:

1)     Make stock sln of Na2HPO4 dibasic (5.25g in 500ml H2O)

2)     Make 2xHBS:

a)     8.0g NaCl

b)    6.5g HEPES (Sigma Cat # H-7006)

c)     10ml Na2HPO4 stock solution

3)     Bring volume close to 500mls. Divide into 3 batches and pH each to 6.95, 7.00, and 7.05. Test each batch using LacZ, DsRed, or GFP to see which pH gives best transfection efficiency.

2.        2M CaCl2:

1)     Add 14.702g of CaCl2?2H2O to 50ml H2O.

2)     F.W. of CaCl2?2H2O = 147.02g. -- > 2M= (2 mole/L)(147.02g/mole)

3)     CaCl2 is from Mallinkrodt, Cat # 4160. It is important you use their CaCl2.

 

 

材料和试剂

1.     适合尺寸的细胞培养板 (Fiosher).

2.     293TPheonix 细胞系

3.     Dulbecco的改良Eagle培养基 (D-MEM) (1X), 液体(高糖) (Invitrogen)

4.     胎牛血清 (Hyclone)

5.     Packaging vector PcmvR8.74

6.     Envelop vector PMD2VSVG

7.     报告基因载体 DsRED, GFP or LacZ

8.     2x HBS

9.     2M CaCl2 (Mallinkrodt Cat # 4160)

 

仪器

1.     细胞培养箱

 

实验步骤

1.     0:

10cm的培养皿中种植4.5×106 293T细胞 (或者是用于逆转录病毒的Pheonix细胞) 37C孵育过夜。你也可以将细胞培养在6cm的培养皿或者6孔板中(细胞种植密度按比例来缩减)。

注意:或者也可以在转染当天种植细胞。见第1天。

2.     1: 转染

注意:如果是在前一天种植细胞,在转染前2h换为含10%胎牛血清的DMEM,然后跳至步骤4

1)     种植293T 细胞(或者是用于逆转录病毒的Pheonix细胞) ,使其在10cm的培养皿中细胞融合度达到50%。我们可以从原来培养皿中的细胞融合度来粗略的估计。例如: 可以将1个融合度为100%10cm培养皿中的细胞种植入210cm培养皿中。也可以将细胞培养在6cm的培养皿或者6孔板中。仅需要使细胞的最终融合度大约为50%. 

2)     37培养箱中孵育细胞3 ~5h,使细胞贴壁。

3)     在进行转染之前,使所有的试剂解冻至室温。

4)     一旦细胞贴壁,准备转染用的DNA混合物。对于每个10cm培养皿,将下列物质加入到5ml聚丙烯管中:

A.     10ug 目的DNA (对于逆转录病毒, 10ug DNA)

B.     6.5ug packaging vector (Pheonix 细胞中已经含有packaging vector,故不需要添加)

C.    3.5ug envelope vector (对于逆转录病毒,5ug envelope vector)

D.    0.1~0.2ug报告基因载体(可以任意选择) – GFP, DsRED, LacZ

E.     0.1% TE水溶液将上述混合物定容至437.5ul

如果使用不同尺寸的培养板,按比例来缩减加液的体积。见下表。

注意:如果你有几块板进行相同的转染,可以配制为一个混合物。然而,为了达到高转染效率需要分别进行每次转染。

注意: DNApackaging vectorenvelope vector的比例对于达到最大病毒滴度是至关重要的。这个比例是由Weissman实验室估计得出的,请尽量按照这个比例来进行转染。

5)     以最高的速度来涡旋混匀DNA混合物,然后加62.5ul 2M CaCl2。继续涡旋,然后向DNA/CaCl2混合物中逐滴滴加500ul 2X HBS (使用1000ul的移液器大约 2 /)

6)     HBS/DNA 混合溶液立即加入细胞中,动作要轻柔,逐滴滴加,使其在培养液中沿着细胞进行扩散。

7)     在几分钟内你就可以在显微镜下观察到细胞表面均匀分布的小的黑色颗粒。

8)     37培养箱中孵育细胞过夜。

 

 

 2xHBS

2M CaCl2

DNA 混合物

DNA:Pack:Env

10cm 培养皿

500ul

62.5ul

437.5ul

10ug:6.5ug:3.5ug

6cm 培养皿

250ul

31ul

219ul

5ug:3.3ug:1.8ug

6 孔板

83ul

10.5ul

73ul

1.5ug:1ug:0.6ug

 

3.     2天:转染后24h

1)     更换为10ml新鲜的含10% 胎牛血清的 DMEM。如果你使用了报告基因载体,现在大概就可以看到转染的效率。 2ug DsRED, 通常可以观察到>75%的转染效率。如果此刻看不到大量的荧光细胞,等到转染后48h再观察。

2)     注意:我们通常是在37培养箱中来包装病毒,虽然有报道说在32条件下孵育,病毒会更稳定。

3)     如果想定量病毒滴度,在感染前一天种植大量细胞。我通常每种病毒种植310cm 培养皿用于3种不同的滴度。

4.     3: 转染后48h

1)     将转染后细胞的上清液分装成所需要的体积,-80冷冻以杀死上清液中的所有细胞 。或者,可以使用45um的过滤器过滤去除上清液中的细胞;如果有大量的漂浮细胞也可以先离心上清液。然而,你需要在使用病毒之前冻结或者过滤上清液以完全去除任何细胞污染的可能性。

2)     将病毒保存于-80

如果你要滴定或者感染目的细胞,请完成接了下来的步骤:

3)     10cm培养皿的细胞中(10ml培养液)加入10ul 1000×凝聚胺(1000× = 5-8ug/ml)。为了滴定病毒,每个皿的细胞中加入0.1ul, 1ul, 10ul病毒上清液,37孵育过夜。

5.     4天:移去病毒上清液

1)     感染后24 h,更换新鲜培养液,37 孵育过夜。

6.     第五天: 感染后24-48h

1)     此时细胞可以用于生物化学方面的检测 (例如,选用合适的抗生素开始选择性培养)。真正的反转录和整合发生在24-36小时,这有赖于细胞的生长周期。

2)     像平常一样培养细胞。

 

配液

2xHBS 用于磷酸钙共沉淀转染:

1.     配制Na2HPO4储存溶液(5.25g 溶于500ml H2O)

2.     配制2xHBS:

1)     8.0g NaCl

2)     6.5g HEPES (Sigma Cat # H-7006)

3)     10ml Na2HPO4 储存溶液

3.     定容至500ml,然后将溶液均分成3 份,分别调pH 6.95, 7.00, 7.05. 使用LacZ, DsRed, GFP 来检测哪个PH条件下的转染效率最高。

1)     2M CaCl2:

A.     50ml H2O中加入14.702g CaCl2?2H2O

B.     CaCl2?2H2OF.W.147.02g. → 2M= (2 mol/L)(147.02g/mol)

C.    CaCl2 购买于 Mallinkrodt, Cat # 4160. 使用它们的CaCl2非常重要。

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How to cite this protocol: Huang, Y. (2011). Lentivirus and Retrovirus Transfection. Bio-protocol Bio101: e38. DOI: 10.21769/BioProtoc.38; Full Text



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1/10/2015 8:04:48 PM  

Syed Raza
Unversity of Utah

Hi i have a question:
How to know what ratio should I took of DNA/envelope/packaging vector for my target vector of 14kb which is a second generation compatible. I tried with 12.5:12.5:5 (DNA:Packaging:Envelop) for 150mm dish, and importanly, can i take the same ratio (12.5:12.5:5) and divide it for 6 well plate as the surface area of the 150mm plate is approx 152 & of 6 well plate is 9.5. If not then how this ratio has to be decided. for e.g. how in your protocol it is adjusted to 167.5ul (total) of 1.5μg:1μg:0.6μg. please kindly explain. My Vector is of 14 kb. Thanks

Syed
Syedshadabraza@gmail.com

1/10/2015 8:05:28 PM  

Syed Raza
Unversity of Utah

my mail id is syedshadabraza@gmail.com

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9/23/2013 11:04:22 AM  

weicheng chang
UC berkeley

Hi:
May I ask a question about the difference between Lenti virus and Retrovirus? I want to pack retrovirus via a viral plasmid backbone "FUGW" which is designed for Lenti virus package. Do you think it will be work or not? Are there any difference sequence design for Lenti and retro virus plasmid backbone? Thanks very much.

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5/31/2011 9:18:24 PM  

Anonymous Lin
Test institute

If i get 83% transfection of 293T cells by Flow what is my titer?

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