The overall goal of this procedure is to learn about lentiviral packaging. A five day protocol. On the first day, 2 9 3, TN producer cells are plated at a calculated density.
On the second day, cells are transfected with the lenti vector construct of choice containing the gene of interest and viral packaging plasmid mix. On day four, the viral sate is harvested and concentrated by adding PEG virus precipitation solution. The final steps of the procedure are to harvest the PEG concentrated virus, aliquot the virus, and infect cells with the concentrated virus for viral tittering.
Ultimately, quantitative PCR of stably integrated genes is used to calculate the number of stable integrated constructs. The main advantage of this technique over existing methods like plasma transfection or retroviral transduction, is that FBI's optimized lentivirus packaging protocol produces high titer functionally active virus, which enables efficient delivery into almost any mammalian cell, including difficult to transfect cells like stem cells and non dividing primary cells. Generally, individuals new to this method will struggle because they are not familiar with the small protocol details, which are essential for successful production of high titer virus.
Visual demonstration of this method is useful as the cell density visualized through a microscope during transfection determines the efficiency of viral packaging, which determines the final viral titer. In cases where applicable fluorescent reporter expression determines the efficiency of transfection, which has a significant impact on the final viral titer. Additionally, visual demonstration of this method is helpful for researchers to familiarize themselves with subtle yet important parts of the protocol, such as best practices to maintain sterility during the entire process, and how to avoid including cell debris in the harvested virus.
Efficient lentiviral packaging relies on three components. First, the lentiviral expression vector must contain some of the elements required for virus packaging and stable integration, as well as elements for expression of the gene of interest. Short hairpin, RNA, micro RNA, or a reporter cassette.
Second, the lentiviral packaging plasmids must provide the proteins essential for packaging an RNA copy of the expression construct into recombinant pseudo viral particles and subsequent reverse transcription and virus integration upon infection. Thirdly, 2 9 3 TN producer cells must be transiently cot transected with the expression and packaging vectors. These cells produce high titers pseudo viral particles to be harvested from the culture media.
Ultimately, successful transduction of the target cells mostly depends on the number of viral particles per cell for a given cell type. In other words, the multiplicity of infection begin by growing up 2 9 3 TN cells. In 15 centimeter cell culture plates for 18 to 24 hours to obtain 60 to 80%co fluency.
At the time of transfection, each plate will yield approximately 14 million cells to increase virus yield. More than one plate can be used the next day when the 2 9 3 TN cells are 60 to 80%confluent, they're ready for transfection. For each plate of cells, add 1.6 milliliters, serum free DMEM to a 15 milliliter falcon tube.
Then add 22.5 micrograms of ppac H one or Ppac F1 and 4.5 micrograms of your lenti vector Construct to the DMEM and mix by pipetting up and down. Next, add 55 microliters of pure affection into the same tube and vortex for 10 seconds, followed by a 15 minute room Temperature incubation. After 15 minutes, add one tube into each culture plate.
Dropwise then gently swirl the plate, then incubate the cells for 48 hours at 37 degrees Celsius with 5%carbon dioxide after 48 hours. If the lenti vector construct expresses a fluorescent gene like GFP, check the cells under a microscope, a successful transfection will yield 50 to more than 90%green fluorescent cells now collect the cell culture supernatant into a 50 milliliter conical tube. The supernatant contains the infectious pseudo viral particles.
Any cells pulled off the plate will not negatively affect the production of virus. To remove debris centrifuge, the viral supernatant at 1500 G for 15 minutes at room temperature. Then without disturbing the pellet, collect the viral supernatant being sure to leave a buffer of about 500 microliters of supernatant and proceed to the next step, which is to concentrate the viral particles 72 hours post transfection.
An optional second collection of the viral supernat may be done using the same technique after the viral sate is collected. Concentrated by adding one volume of ice cold peg virus precipitation solution to every four volumes of supernatant in a 50 milliliter polypropylene centrifuge tube. Invert the solution to mix it.
Never use a vortex, then store the solution without agitation at four degrees Celsius for up to 96 hours. After a minimum of 12 hours of refrigeration centrifuge the mixture at 1500 G for 30 minutes at four degrees Celsius. After the centrifugation, the peggo precipitated lentiviral particles will appear as a beige or white pellet at the bottom of the tube.
Now, remove all traces of fluid by careful aspiration without disturbing the precipitated lentiviral particles in the pellet. Alternatively, remove most of the supra natin and resuspend the pellet in remaining supinate. Then transfer the suspension to a sterile two milliliter einor tube and centrifuge the tube in a cold micro fuge for two minutes.
At 14, 000 RPM pipette away the remaining supernatant and resuspend the lentiviral particle pellet in ice, cold, sterile buffer at one 10th to one 100th. The original volume carefully pipette the pellet into solution without creating any bubbles, keeping it cold at all times. Make sure the pellet is completely resuspended.
Lastly, make 15 to 25 microliter aliquots ink, sterile cryogenic vials on ice and store the vials at negative 70 degrees Celsius. A successful packaging run will yield 10 million to 1000 million IFUs per milliliter for virus transduction in a 24 well culture plate plate, 50, 000 cells per well and grow them overnight at their specified culture conditions. The cells should be between 50 to 70%confluent for transduction for tittering purposes.
HT 10 80 cells can be used as an easy to transduce control cell line the next day aspirate the media from the cells. Then combine the culture medium with concentrated duck solution. So the final concentration of trans ducks is one x, add one half milliliter of the transduced to each well with target cells at the correct density of 50 to 70%Co fluency add virus to each well at the following dilutions, one to 100, one to 10, and straight one-to-one dilution.
The virus can remain in contact with the target cells for up to 72 hours without changing the medium. After 72 hours, the provirus will have stably integrated into the host genome and be expressing the gene of interest. If present fluorescent marker expression can be used to estimate the transduction efficiency with different virus dilutions, then the number of infectious units can be quantified by QPCR.
Once the virus titer is known, calculate the appropriate volume of virus to achieve the desired multiplicity of infection for other target cells. Measuring the titer and controlling MOI ensures consistency between experiments and different viral preps. The virus is now ready for use in additional experimentation.
The starting density of the 2 9 3 TN cells was critical for successful viral packaging on the day of transfection. These 2 9 3 TN cells were between 60 and 80%confluent. 24 hours after a successful transfection with a lenti vector expressing GFP, it was found that at least 90%of the 2 9 3 TN cells showed green fluorescence.
72 hours post transduction GFP continued to express in the HT 10 80 host cells. This indicates that the lentiviral construct stably integrated into the host genome transducing the same host cells at tenfold dilution proportionately decreased the expression of GFP. Because the relationship between viral titer and lentiviral mediated gene expression is directly proportional.
Once mastered, this technique can be done over a course of five days at approximately one to two hours per day if it is performed properly. After watching this video, you should have a good understanding of how to package functionally infectious viral particles efficiently and safely and successfully transduced cells with the virus. Don't forget that working with lentivirus can be hazardous and precautions such as working in BSL two tissue culture hood.
Wearing lab coat and gloves should always be taken while performing this procedure.
