Question

Explain a method to deliver your recombinant DNA to HEK293 culture cells. Explain the steps of your method.

Answer 1

One of the convenient method to deliever a recombinant DNA in HEK293 cells, is using adeno-associated virus (AAV) as a vector.

For the first step, three types of plasmids are required by AAV to transfect HEK293 cells. The RepCap plasmid is needed to convert double-stranded intermediate into single-stranded RNA. It is also required to assemble viral particles inside the capsid. Helper plasmid assists in replication of viral particles and transfer plasmid encodes the gene of interest that is transferred into virus from HEK293 cells via transfection.

All these plasmids are co-transfected with HEK293 cells for 2 to 5 days, after which cells are harvested and transferred to a cylindrical tube. Then, polyethylene glycol (PEG) is added to this tube and centrifugation is done. Supernatant is formed, to which benzonase solution is added.

Benzonase acts as endonuclease that breaksdown the DNA particles remaining in the supernatant. Then, iodixanol solution is added to the supernatant for futher purification. Afterwards, viral particles are harvested and collected and their quantification is done by qPCR. These viruses are used to infect target cells and their expression are monitored carefully.