Paul.xu at 04:08, 21 November 2013

2013-11-21T04:08:35Z

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== Production of Cre VLP using 293E ==

1. Plate 3.3 × 106 293E cells in a 10-cm dish, add 10 mL of DMEM containing 10%  FBS and G418 (50 μg/mL)  24 h before transfection. 

2. On the day of transfection, replace the culture medium with 10 mL of fresh medium. 

3. Transfect 15 μg of DNA. 

'''Tube A''': Add to 500 μl of DMEM (no serum) 

 

9 μg of pCMV5-Gag-Cre-oriP (clone id is 1400, pCMV5-Gag-Cre)

(for FPL use 1445 pCMV5-Gag-NLS-Flp-WPRE-oriP to replace 1400, pCMV5-Gag-Cre)

5.5 μg of pCMV-VSV-G (505 pCMV-VSVG)

0.5 μg CMV-GFP plasmid (507 pSMPUW-CMV-GFP) 

 

'''Tube B''': 45 μl of GeneExpressoMAX to 500 μl of DMEM (no serum) 

Mix two tubes, incubate 20 min. 

4. Add to cells. 

5. 48–72 h later, collect medium. 

6. Spin 3000 rpm for 10 min at 4 °C. 

'''Steps 7~10 except ultracentrifugation should be done inside a biosafety hood to avoid bacterial contamination:''' 

7.  add filter sterilized DNase1 (10 units/mL) to clarified supernatants, incubate  for 30 min at room temperature. 

8. Filter through a 0.45-μm filter. VLP in medium can be used immediate for transduction or stored at −80 °C.

9. To concentrate VLP: transfer medium to ethanol-sterilized tubes and spin at 28000 rpm for 2.5 hours on a 30 % sucrose cushion. 

30 % sucrose/HEPES buffer (2 mM HEPES, pH 7.4, 125 mM NaCl, 0.9 mM CaCl2, 0.5 mM MgCl2) 

Gently remove supernatant and sucrose, add 1~5 ml of filter-sterlized 15% trehalose/10% glycerol in PBS. Transfer pellet to sterilized tubes, incubate in cold room to dissolve VLP from pellet. 

 [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3502365/ http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3502365/] 

10. Aliquote VLP, freeze on dry ice and store at −80 °C.

11. Assay VLP titer using reporter cell line.

== Reporter Assay ==

 

1. Transfect 293 (T or E) cells  with 1522 CMV-Brainbow-2.1-R or 1053 pLVX-loxP-sfGFP-loxP-TagRFP. 

2. Add VLP

3. Observe color change in the next few days

 

 

[[Category:Protocols]]

Virus-like particle Production - Revision history

Paul.xu at 04:08, 21 November 2013