TITLE: Nanoblade production
AUTHORS: maxime.smits , Simone Giovannozzi, Louise Medaer

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Nanoblades are virus-like particles that are able to transiently deliver a Cas9 protein together with a gRNA, without integration of DNA in the genome.</div></div>

[STEPS]
?. [DAY 0]
SEEDING Petri-dishes (PD)
?. [DAY 0]
Seed HEK293T cells in a 10 cm PD in DMEM 2% FCS + 500 µL Gentamycin
?. [DAY 0]
Incubate on 37 °C at 5% CO2
?. [DAY 1]
TRANSFECTION of transfer, packaging, and envelop plasmids
?. [DAY 1]
Before starting, measure the DNA concentration and the purity of the plasmids in the molecular lab.
?. [DAY 1]
Make a DNA mixture in an 1,5 mL tube of the following plasmids: 1 PD5 PDGAG-POL (MLV-gag-pol Els Verhoeyen**)6,38 µG31,9 µGBIC-Cas9 (BIC-SpCas9 Els Verhoeyen**)6,38 µG31,9 µGCrizi-sgRNA* (Crizi Els Verhoeyen**)6,38 µG31,9 µGVSV-G (VSVG Els Verhoeyen**)1,59 µG7,95 µGBaevR (hCMV-BaevR Less ClaI-HpaI**)1,59 µG7,95 µG
1 PD5 PDGAG-POL (MLV-gag-pol Els Verhoeyen**)6,38 µG31,9 µGBIC-Cas9 (BIC-SpCas9 Els Verhoeyen**)6,38 µG31,9 µGCrizi-sgRNA* (Crizi Els Verhoeyen**)6,38 µG31,9 µGVSV-G (VSVG Els Verhoeyen**)1,59 µG7,95 µGBaevR (hCMV-BaevR Less ClaI-HpaI**)1,59 µG7,95 µG
?. [DAY 1]
Write down the total volume of the DNA mixture
?. [DAY 1]
Transfer the DNA mixture in a 50 mL Falcon tube in the vector lab, under the flow
?. [DAY 1]
Add autoclaved PBS to the mixture with a final volume of 557 µL (1 PD) or 2785 µL (5 PD)
?. [DAY 1]
Dilute the linear PEI (323 mg/L) with autoclaved PBS:1 PD5 PDLinear PEI245,5 µL1227,5 µLPBS311,0 µL1555,0 µL
1 PD5 PDLinear PEI245,5 µL1227,5 µLPBS311,0 µL1555,0 µL
?. [DAY 1]
Add the diluted linear PEI to the DNA mixtures GENTLY, and incubate for 5 to 10 minutes on RT
?. [DAY 1]
Add 5 mL (1 PD) or 25 mL (5 PD) of cold medium (DMEM 2% FCS + 500 µL Gentamycin or OptiMem + 500 µL Gentamycin) to the DNA-PEI mixtures GENTLY and mix
?. [DAY 1]
Check the seeded HEK293T cells under the microscope for confluency
?. [DAY 1]
Remove the medium on the HEK293T cells and add the transfection medium. Normally there is enough for 6 mL of medium per PD
?. [DAY 1]
Incubate on 37 °C at 5% CO2
?. [DAY 2]
REMOVING TRANSFECTION MEDIUM
?. [DAY 2]
Remove the Transfection medium and replace it with RT medium. 5 mL per PD should be sufficient
?. [DAY 1]
Leave the medium flask at RT
?. [DAY 2]
Incubate on 37 °C at 5% CO2
?. [DAY 3]
1st HARVEST
?. [DAY 3]
Collect the supernatant from the PD using a 0,45 µM syringe and store at 4 °C
?. [DAY 2]
Also store the medium flask at 37 °C
?. [DAY 3]
Add Pre-warmed medium to the cells.
?. [DAY 3]
Incubate on 37 °C at 5% CO2
?. [DAY 4]
2nd HARVEST
?. [DAY 4]
Collect the supernatant from the PD using a 0,45 µM syringe and store at 4 °C
?. [DAY 4]
Desinfect the PD by spraying 70% Ethanol or bleach on the cells, before throwing them in the yellow bin
?. [DAY 4]
The supernatant of both harvest days will be concentrated via vivaspin 1. First wash the vivaspin membrane by adding 10 mL of PBS and spinning down at 3000g for 3 minutes2. Add the supernatant to the vivaspin membrane and spin down at 3000g, until there is about 0,5 - 1,0 mL remaining
?. [DAY 4]
Nanoblades can be stored at -80 °C