What does transduction efficiency mean?
Measure Transduction Efficiencies Optimization of cellular transfection and transduction includes choosing a protocol, determining the appropriate mass of plasmid/virus, and evaluating the optimum time after transfection/transduction for the best expression of the construct of interest.
What is lentivirus transduction?
Lentiviral transduction is an efficient method for the delivery of transgenes to mammalian cells and unifies the ease of use and speed of transient transfection with the robust expression of stable cell lines.
How does lentivirus transduction work?
Adenoviral Transduction Adenoviral vectors have proven to be a very successful transduction tool in many eukaryotic cell types, such as human and rodent cells. Besides dividing cell lines, this method gives access to difficult-to-transfect cells, such as primary cells.
How long does it take to transfect?
Post-transfection Incubation Time In general, maximal protein expression occurs 48 hours post-transfection. The time point for optimal gene expression can be determined by varying post-transfection incubation times from 4 to 72 hours.
Can you transduce cells twice?
It is a routine practice in our lab (human and murine cells) and we have no problem with this type of protocol. You can also do the transuction in two times if you want an offset expression of the two transgenes, it depends of the experimental purposes.
How do you measure transduction efficiency?
To determine transduction efficiencies, a total of 5 × 104 cells (adherent cells) or 1 × 105 cells (suspension cells) per well in 24-well plates were inoculated with serial dilutions of the virus supernatants overnight.
Which transduction is more effective?
Most recently, Zeira et al. revealed that gene delivery in muscle by the laser beam gene transduction (LBTS) is more efficient and safe compared to gene delivery by EP in the same muscle under the same conditions.
What are the two main functions of Lentivirus recombination?
The Lentivirus – The Recombinant Lentivirus System
| Molecular | Function |
|---|---|
| 1. Cis-acting | |
| LTRs | Contain sequences required for viral gene expression, reverse transcription, and integration |
| ψ | Required for packaging of the genomic transfer RNA |
| RRE | Rev response element. Required for processing and transport of viral RNAs |
How is the shRNA used in lentiviral transduction?
In this instance, the shRNA is first packaged into the lentiviral vector, and is then used to transfect HEK293 cells. These transfected cells are allowed to incubate for ~3 days, which permits the lentivirus to replicate and produce lentiviral particles that are then harvested, titered, and used to transduce target cells.
What is the transfection efficiency of lentiviral cells?
We routinely achieve >99% transfection efficiency in Lenti-X 293T cells when using our optimized reagents. The Lenti-X Maxi Purification Kit will remove transduction inhibitors, and result in up to a 10-fold concentration of virus, depending on the starting supernatant volume.
How are HEK293 cells seeded for lentiviral transduction?
For the transfection experiment, HEK293 cells should be seeded at about 5*10^6 cells/10cm dish or in such a way that the cells are about 40-50% confluent the next morning (day of the transfection). The next step is to package the lentiviral vector plasmid, along with viral packaging vectors, into liposomes for delivery into HEK293 cells.
Which is the best packaging system for lentiviral transduction?
Our pLVX vectors are optimized for both titer and expression, and will produce higher titers than other vectors. The Lenti-X Packaging System is likely to produce the highest titer for any HIV-1-based lentiviral vector.