Editing Virus-like particle Production (section)

== Production of Cre VLP using 293E <br>  ==

1. Plate 3.3 × 10<sup>6</sup> 293E cells in a 10-cm dish, add 10 mL of DMEM containing 10%&nbsp; FBS and G418 (50 μg/mL)&nbsp; 24 h before transfection.<br> 

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

3. Transfect 15 μg of DNA.<br> 

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

<br> 

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)<br> 

<br> 

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

Mix two tubes, incubate 20 min.<br> 

4. Add to cells.<br> 

5. 48–72 h later, collect medium.<br> 

6. Spin 3000 rpm for 10 min at 4 °C. <br> 

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

7.&nbsp; add filter sterilized DNase1 (10 units/mL) to clarified supernatants, incubate&nbsp; for 30 min at room temperature.<br> 

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&nbsp;% sucrose cushion. <br>

30 % sucrose/HEPES buffer (2 mM HEPES, pH 7.4, 125 mM NaCl, 0.9 mM CaCl<sub>2</sub>, 0.5 mM MgCl<sub>2</sub>)<br><br>

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.<br> 

&nbsp;[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3502365/ http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3502365/] <br> 

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

11. Assay VLP titer using reporter cell line.