Transfection of Axol Cortical Neural Stem Cells (hNSCs)

Background:

Transfection is the process of deliberately introducing nucleic acids into cells. For some applications of transfection, it is sufficient if the transfected genetic material in only transiently expressed. Since the DNA introduced in the process is usually not integrated into the nuclear genome, the foreign DNA will be diluted through mitosis or degraded. If it is desired that the transfected gene actually remain in the genome of the cell and its daughter cells, a stable transfection must occur.

There are several common methods for the process of transfection such as:-

• Chemical-based methods (e.g. Polymers, liposomes, etc.)
• Non-chemical methods (e.g. Electroporation, Optical etc.)
• Particle-based methods (e.g. Magnet assisted)
• Viral methods (e.g. Adenoviral vectors)
• Other hybrid methods (e.g. Nucleofection)

The purpose of this technical application note is to investigate the suitability of a lipid-based reagent (Axol ReadyFect™) for use with Axol Cortical Neural Stem Cells.

Axol ReadyFect™ is a lipid-based transfection reagent based on Triggered Endosomal Escape technology which combines and exploits the properties of cationic lipids and polymers to provide a transfection method optimized for use with our hNPCs. 

The optimizations mean that Axol ReadyFectTM:-
• has minimal hNSC toxicity
• does not impact differentiation
• does not impact phenotype potential
• achieves efficient DNA delivery into the cells
 

Protocol:

1. DNA solution. Dilute 0.125 µg to 10 µg of DNA in 25 µL to 350 µL (Table 1) of PBS.
2. Axol ReadyFectTM solution. Dilute 0.375 µL to 30 µL of Axol ReadyFectTM (Table 1) in PBS.
3. Add DNA solution to the Axol ReadyFectTM solution, mix gently by vortexing slowly or pipetting up and down 4-5 times. Incubate the mixture for 20 mins at room temperature. The diluted solutions should be combined within 5 minutes.
4. Add the mixture to Axol Cortical Neural Stem Cells (growing in Complete Axol Neural Maintenance Medium) dropwise and homogenize by gently rocking the plate side to side to ensure a uniform distribution of the mixture.
5. Incubate the cells at 37°C, 5% CO2.
6. After 4-6 hr of incubation, replace the transfection medium with fresh Complete Axol Neural Maintenance Medium.
7. Incubate the cells at 37°C, 5% CO2.
8. Evaluate your transgene expression 24 to 72 hr post-transfection.
Odoo image and text block

Results:

Odoo image and text block

Axol ReadyFectTM leads to 30-40% transfection efficiency. The image demonstrates transfection results using 3 µL of Axol ReadyFectTM/ 1 µg of a GFP-encoding vector (pVectOZ-GFP). GFP expression was evaluated 2 days post-transfection. 9 lipids (NL-1 to NL-9) were tested using 3 ratios. Axol ReadyFectTM (NL-7) gave the best fluorescence intensity of the lipid-based transfection reagents tested. 

Table 1: Suggested amounts of DNA and reagent depending on your cell culture format

Cell Culture Vessel DNA Quantity Dilution Volume Axol ReadyFectTM Volume Dilution Volume Final Transfection Volume
96 Well Plate 0.125 ug 25 uL 0.375 uL 25 uL 200 uL
24 Well Plate 0.15 ug 50 uL 1.5 uL 50 uL 500 uL
6 Well Plate 2 ug 100 uL 6 uL 100 uL 2 mL
60mm Dish 3 ug 150 uL 9 uL 150 uL 4 mL
T-75 Flask 10 ug 350 uL 30 uL 350 uL 10 mL

Conclusion

  • Axol ReadyFect TM   leads to 30-40% transfection efficiency.