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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, 20501 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. 293T and Pheonix cell line
  2. 1x Dulbecco's modified eagle medium (DMEM) (high glucose, liquid) (Life Technologies, InvitrogenTM)
  3. Fetal bovine serum (FBS) (Hyclone)
  4. Na2HPO4
  5. NaCl
  6. TE
  7. HEPES (Sigma-Aldrich, catalog number: H7006 )
  8. Packaging vector PcmvR8.74
  9. Envelop vector PMD2VSVG
  10. Reporter vector DsRED, GFP or LacZ
  11. 2x HBS (see Recipes)
  12. 2 M CaCl2 (Covidien, MallinkrodtTM, catalog number: 4160) (see Recipes)

Equipment

  1. Tissue culture incubator
  2. Tissue culture plates of the appropriate size (Thermo Fisher Scientific)
  3. 6 cm or 6 well plates
  4. Microscope
  5. 0.45 μm filter
  6. p1000 pipettor

Procedure

  1. Day 0
    Plate 4.5 x 106 293T (or Pheonix for retrovirus) on 10 cm plate. Incubate overnight at 37 °C . You can also scale down to 6 cm 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 2 h prior to transfection and skip to step 4.
    1. Plate 293T (or Pheonix for retrovirus) at ~50% confluence on 10 cm TC plates. I calculate it roughly from the original plate confluence. Example: I will plate 2 x 10 cm plates from 100% confluence 10 cm plate. I have scaled down to 6 cm and 6 well plates. Just keep final confluence of cells at roughly 50%.
    2. Incubate plates in 37 °C incubator for ~3 to 5 h 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 10 cm plate, add the following to 5 ml polypropylene tube:
      1. 10 μg DNA of interested (for retrovirus, 10 μg DNA)
      2. 6.5 μg packaging vector (packaging vector already in Pheonix, so no need to add)
      3. 3.5 μg envelope vector (for retrovirus, 5 μg envelope vector)
      4. 0.1 to 0.2 μg of reporter vector (optional) - GFP, DsRED, or LacZ
      5. Bring mixture up to 437.5 μl with 0.1% TE in H2O
      If you use different size plates, just scale down. See table below.
      Notes: 
      1. 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.
      2. The ratio of DNA: packaging vector: envelope vector is crucial for max viral titer. The ratio provided here was determined by the Weissman Lab. Please keep to this ratio as much as you can.
    5. Vortex the DNA mixes on highest speed setting and add 62.5 μl of 2 M CaCl2. While still vortexing, add 500 μl 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.


      2x HBS
      2 M CaCl2
      DNA mix
      DNA: Pack: Env
      10 cm plates
      500 μl
      62.5 μl
      437.5 μl
      10 μg: 6.5 μg: 3.5 μg
      6 cm plates
      250 μl
      31 μl
      219 μl
      5 μg: 3.3 μg: 1.8 μg
      6 wells plate
      83 μl
      10.5 μl
      73 μl
      1.5 μg: 1 μg: 0.6 μg

  3. Day 2: 24 h post-transfection
    1. Change medium to 10 ml fresh DMEM 10% FBS. You can generally see the efficiency of your transfection by now if you used a reporter vector. At 0.2 μg of DsRED, I can usually observe >75% transfection efficiency. If you can’t see much fluorescent cells at this time, wait until 48 h post-transfection and observe again.
      Note: We have always packaged our viruses in 37 °C incubator although it has been reported that the virus is more stable if incubation is carried out at 32 °C .
    2. If you want to titer the virus, plate target cells now for infection the next day. I often plate 3 x 10 cm plates per virus for 3 different titrations.

  4. Day 3: 48 h 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 supernatant through a 0.45 μm filter to remove the cells. You can also choose to centrifuge the supernatant first if there are many floating cells. However, you need to either freeze or filter the supernatant before using the virus to completely remove any chances 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 10 μl 1,000x polybrene (1,000x = 5-8 μg/ml) to each 10 cm plate of target cells (10 ml medium). Add 0.1 μl, 1 μl, or 10 μl of virus supernatant to each plate of cells for titering your virus. Incubate at 37 °C overnight.

  5. Day 4: Remove virus supernatant
    1. 24 h post-infection, change to fresh medium. Incubate overnight at 37 °C .

  6. Day 5: 24-48 h post-infection
    1. Cells are now ready to be assayed for any biochemical event of interest (example: start selection with appropriate antibiotic). The actual reverse transcription and integration take place within 24-36 h, depending on cell growth kinetics.
    2. Culture cells as normal.

Recipes

  1. 2x HBS for calcium phosphate coprecipitation transfection
    1. Make stock sln of Na2HPO4 dibasic (5.25 g in 500 ml H2O)
    2. Make 2x HBS
      8.0 g NaCl
      6.5 g HEPES
      10 ml Na2HPO4 stock solution
    3. Bring volume close to 500 ml. 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. 2 M CaCl2
    1. Add 14.702 g of CaCl2·2H2O to 50 ml H2O. 
    2. F.W. of CaCl2·2H2O = 147.02 g. -- > 2 M= (2 mol/L) (147.02 g/mol)
    3. CaCl2 is from Mallinkrodt (catalog number: 4160). It is important you use their CaCl2.

font-size:10pt; font-family:Arial;" lang ="EN-US">材料和试剂

 

1.      class ="Apple-converted-space">  lang ="EN-US">具有适当大小的组织培养板( Fiosher )。

2.      class ="Apple-converted-space">  lang ="EN-US"> 293T和  Pheonix 空间">  单元格

3.      class ="Apple-converted-space">  Dulbecco改良的Eagle培养基(D-MEM)(1X),液体(高葡萄糖)(Invitrogen)

4.      class ="Apple-converted-space">  lang ="EN-US">胎牛血清( Hyclone

5.      class ="Apple-converted-space">  lang ="EN-US">包装矢量PcmvR8.74

6.      class ="Apple-converted-space">  lang ="EN-US">信封向量  PMD2VSVG

7.      class ="Apple-converted-space">  lang ="EN-US">记者向量  DsRED ,GFP或  LacZ

8.      class ="Apple-converted-space">  lang ="EN-US"> 2x HBS

9.      class ="Apple-converted-space">  lang ="EN-US"> 2M CaCl 2   Mallinkrodt   Cat#4160)

 

设备

 

1.      class ="Apple-converted-space">  lang ="EN-US">组织培养箱

 

程序

 

1.      class ="Apple-converted-space">  lang ="EN-US">第0天:

Plate 4.5x10 6   293T(或 Pheonix   转换空间">  10cm     37°C孵育一夜。您还可以缩小到 6cm   或6孔板。 span>

注意:或者,您可以在 transfection 。见第1天。

2.      class ="Apple-converted-space">  lang ="EN-US">第1天:TRANSFECTION

注意:如果您在前一天接种细胞,则在   并跳到第4步。

1)     -space">  Plate 293T(或  Pheonix 转换空间"></span>用于逆转录病毒)在10cm TC板上在约50%汇合。我从原始板大致计算 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 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) 

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)

合流。例如,我将从1 100%汇合10厘米板2 X 10厘米板。我已经缩小到6厘米和6孔板。

2)     -space">  中培养平板37°C   孵育器约3至5小时以允许细胞附着。

3)     -space">  在进行 转染之前,将所有试剂溶解至室温。

4)     -space">  一旦细胞附着,准备 DNA   混合  transfection 。对于每个10cm平板,向5ml聚丙烯管中加入以下物质:

a)       "Apple-converted-space"> 10μg DNA   逆转录病毒,10μg  DNA)

b)      -converted-space"> 6.5μg包装矢量(包装矢量已位于  Pheonix 无需添加)

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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  

Yuanqing Lin
Test institute

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

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