How do you increase lentivirus transduction efficiency?
10 best practices for a good transduction efficiency
- 1- Know your cells.
- 2- Check the quality of your lentiviral vector batch.
- 3- Optimize the transgene expression cassette.
- 4- Make sure your titer is in TU/ml.
- 5- Follow storage and thawing requirements.
- 6- Monitor cells sensitivity towards transduction enhancers.
What is transduction efficiency?
For lentiviral constructs with a fluorescent marker or antibiotic resistance marker, transduction efficiency (i.e., % infected cells) can be determined from the fraction of fluorescent or antibiotic resistant cells in the population.
Why is polybrene used for transduction?
Polybrene is a cationic polymer that can greatly enhance the efficiency of the retroviral or lentiviral infection to the mammalian cells. It acts to neutralize the charge repulsion between virions and the cell surface, thereby increasing infection efficiency.
What is the optimal moi?
According to Table 1, the optimal MOI to use is 2. So, if you purchase 50 microliters of viral particles and the titer is 108 TU/ml, then you should have a total of 0.05 ml x 108 particles ml-1 = 5 x 106 particles.
How does Polybrene increase the efficiency of gene transfer?
Cationic polymers, such as polybrene and protamine sulfate, are typically used to increase the efficiency of retrovirus-mediated gene transfer, however, the mechanism of their enhancement of transduction has remained unclear.
How does Mois affect the efficiency of transduction?
At lower MOIs, only minimal differences between the varied transduction methods can be observed. At higher MOIs, a more dramatic effect is seen with fibronection producing an approximately 50% increase in surviving cells over polybrene, while spinoculation generated approximately a 5-fold increase in surviving cells over polybrene. Figure 1.
How is Polybrene used as a transfection reagent?
Polybrene with a DMSO shock is also used to mediate DNA transfer into a variety of cell types, such as CHO, chicken embryo fibroblasts, NINH-3T3 and Myeloid cells. Recombinant retroviral stocks are prepared by adding 5mls of growth medium with 5% serum to a near confluent monolayer of transfected retroviral packaging cells in a 100mm plate.
How does Polybrene increase the efficiency of retroviral infection?
It acts to neutralize the charge repulsion between virions and the cell surface, thereby increasing infection efficiency. The efficiency of retroviral infection is enhanced significantly, 100 to 1,000 fold in some cells, by including polybrene during the infection.