Plasmid Purification and Cell Culture
1. Prepare high quality plasmid DNA.
2.18 - 24 hours prior to transfection, plate 2.5 x 106 of 293T cells on a 10cm dish and incubate at 37 ℃ overnight.Cells should reach 65-70% confluency within 24 hours.
Transfection into 293T Cells
3. Add transfer vector and packaging plasmids into the Opti-MEM. Mix by
4. Add transfection reagent into the same tube. Vortex for 10 seconds.
5. Incubate the mixture at room temperature for 15 minutes.
6. Add the mixture drop-wise to the dish, and swirl to disperse evenly throughout the plate.Return the dish to the cell culture incubator at 37°C.
Harvest Viral Supernatant
7. After 12-18 hours incubation, change the culture medium and continue to incubate the plate for 48 hours.
8. Transfer the cell culture supernatant to a 15mL centrifuge tube. Centrifuge at 3000 x g for 15 mins and filter the supernatant through a syringe filter (0.45 micron). Transfer the viral supernatant into a new tube.
9. The viral particles are ready to be used. They can be stored at 4 ℃ for 2 weeks or aliquot and store at -80℃ for long-term.
10. Plate 50 000 target cells per well in a 24 well plate to 50% confluency upon transduction.
11. Remove medium from wells and add appropriate amount of Lentiviral particles, culture medium, polybrene (Optional). Gently swirl the plate to mix.(Optional: Add increasing amounts of virus to different wells at varying MOIs (5, 10 and 20, etc.) to optimize the transduction).
12. 72 hours post transduction, the viral genome will be integrated into the host cell genome.Harvest the cells and perform qRT-PCR or Western blot or flow cytometry.
Lentiviral transfer plasmid contained the gene of your interest. The transgene sequence is flanked by long terminal repeat (LTR) sequences, which facilitate integration of the transfer plasmid sequences into the host genome. Sino Biolicial transfer plasmids are the optimized vectors which contained specific elements to improve transgene expression, virus titer, biosafety. In addition to yielding high expression levels, the promotor for transgene expression is an enhancer CMV promotor.cPPT element enhances transduction efficiency and WPRE enhances expression of your transgene, and increases viral titer. For safety reasons, transfer plasmid is replication incompetent and contains an additional deletion in the 3'LTR, rendering the virus "self-inactivating" (SIN) after integration.
The VSV-G envelope plasmid contains the G protein of the Vesicular Stomatitis Virus (VSV-G) envelope gene. Unlike the HIV envelope, the VSV-G envelope has a broad cell host range extending the cell types that can be transduced by VSV-G-expressing lentivirus.The REV plasmid contains the regulatory protein rev that is required for HIV replication.
The packaging plasmid contains contains the structural (gag), and replication (pol) genes which code for some of the proteins required to produce the lentivirus. It also encodes the viral env gene, which encodes the envelope protein that defines the tropism (i.e.the range of infectable cells).
The envelope plasmids and packaging plasmids provide all of the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant pseudoviral particles.