Paul.xu at 00:30, 17 November 2013

2013-11-17T00:30:23Z

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1. Seed 3.3 × 106 293E cells on a 10-cm dish  with 10 mL of DMEM containing 10% FBS and  50 μg/mL of G148.

2. On the day of transfection, change medium.

3. 2 hours after medium change, transfect cells with 15 μg of DNA:

 

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) 

 

45 μl transfection reagent 

 

4. Collect conditioned medium  on '''day 3''', replace with 10 ml fresh medium per 10 cm plate, collect conditioned medium on '''day 5 or 6'''. Store medium in an autoclaved container in cold room. 

5. Spin at 2200 × g for 10 min at 4 °C.

6. Optional: treat with DNase1 (10 units/mL) for 30 min at room temperature.

7. Optional: Filter through a 0.45-μm filter.

8. The VLPs can be used for immediate transduction or frozen on dry ice (Add 15 g Trehalose to 100 ml and filter again) and then stored at −80 °C.

9. For concentrated VLP: spin medium at 25000 rpm for 1 hour. Remove medium and add filtered 15% Trehalose in 1XPBS to resuspend pellet.  Store in cold room or 4 oC fridge for 1 ~ 2 days to dissolve the VLP. Filter the VLP with 0.2 micron filter. 

 

Related Protocol: [[Virus-like_particle_Production|Virus-like particle Production]] 

[[Category:Protocols]]

Cre VLP Production - Revision history

Paul.xu at 00:30, 17 November 2013