Lentiviral (LV) vectors offer unique advantages over other gene delivery systems, namely the ability to integrate transgenes into the genome of both dividing and nondividing cells. Detailed herein is a simple protocol for the production LV vectors, describing the triple transfection of an LV transfer vector and LV helper plasmids into HEK-293 cells, and the subsequent purifi cation of virions from the cellular media.
The current protocol is versatile, and can be easily modifi ed to fi t the specifi c needs of the researcher in order to produce relatively high-titer LV vectors which can be used to transduce a wide variety of cells both in vitro and in vivo.
First of all: some prerequisites
Lentivirus(LV) is an enveloped, plus-strand RNA virusbelonging to the family of retroviridae. LV vectors, derived from the human immunodefi ciency virus (HIV-1), are powerful genetic tools that offer several advantages over other viral vector systems. Principle amongst the advantages of LV vectors is the ability to integrate genetic material into the genome of dividing and nondividing host cells, both in vitro and in vivo. LV vectors can provide effi cient, stable, and long-term gene expression in terminally differentiated cells such as immune cells or neurons. Due to the ability of LV vectors to integrate genetic material into the host genome, progeny produced from the infected cell will harbor the same transgene, an attribute that is exploited in applications such as stem cell therapy.
Further, LV vectors offer a relatively large carrying capacity (8–9 Kb) and exhibit low immunogenicity, making these vectors extremely useful gene deliverytools. Progress in the production of LV vectors has led to the development of replication incompetent LV vectors, which can be produced easily with minimal effort. Replication incompetent LV vectors are produced by replacing all viral genes from the LV genome with a transgene of interest, leaving only the cis-acting elements essential for viral packaging (i.e., long terminal repeats (LTR), tat activation region, Ψ packaging signal, Rev-responsive element, and polypurine tracts). The removed components of the viral genome are supplied in trans by separate plasmids encoding the viral genes necessary for packaging and envelope pseudotyping.
The transfer vector containing the transgene of interest and the separate helper plasmids are co-transfected into a mammalian cell line, and LV vectors can be harvested and purifi ed from the media. Providing the necessary viral genes in separate plasmids greatly reduces the possibility of mobilization and the production of a replication competent virus . Further, this system also affords the ability to easily mix and match glycoproteins from alternative enveloped viruses in order to optimize the expression levels and tropism of LV vectors specifi cally to the desired application.
Here we describe a simple and effi cient protocol for packaging a vesicular stomatitis virusglycoprotein ( VSV-G ) pseudotyped LV vector. The current protocol describes the triple transfectionof a transfer vector and two LV helper plasmids, and the subsequent purifi cation and concentration of LV vectors from cellular media by ultracentrifugation.
This protocol is flexible and can be easily scaled up or down in order to meet the needs of the researcher. Utilizing the current protocol we have generated LV-vectors with titers in the range of 2 × 10 12 viral genomes (vg)/mL, that are capable of transducing neurons within an adult animal in vivo , as well as diffi cult-to-transfect cells in vitro.
Needs of optimum protocols
Plasmids and Cells
- Human Embryonic Kidney 293 T (HEK-293 T) Cells (ATCC, Manassas, VA, USA).
- pNHP packaging vector (Addgene, Cambridge, MA, USA).
- pHEF-VSVG (Addgene, Cambridge, MA, USA).
- pFIN transfer vector (Addgene, Cambridge, MA, USA)
Reagents and Supplies
- Penicillin/Streptomycin (Pen/Strep; 10,000 units/mL).
- Fetal bovine serum (FBS).
- TrypLE Express 1× dissociation reagent (Thermo Fisher Scientifi c, Grand Island, NY, USA).
- 25 kDa linear Polyethylenimine (PEI) (Polysciences, Warrington, PA, USA).
- Dulbecco’s modifi ed Eagle media (DMEM) containing high glucose, glutamine, and sodium pyruvate.
- 1× Dulbecco’s phosphate-buffered saline (DPBS) containing calcium and magnesium.
- 0.22 μm Stericup fi lter unit (EMD Millipore, Billerica, MA, USA).
- 1.5 M NaCl: Add 87.66 g of NaCl to 800 mL of dH 2O, stir until completely dissolved. Adjust to 1 L with dH 2O and fi lter sterilize with a 0.22 μm fi lter unit.
- 0.45 μm Stericup-HV fi lter units (EMD Millipore, Billerica, MA, USA).
- Centricon Plus-70 Centrifugal fi lter units (EMD Millipore, Billerica, MA, USA).
- 38.5 mL conical thinwall polyallomer ultracentrifuge tubes or equivalent (Beckman Coulter, Indianapolis, IN, USA).
- T175 Culture fl asks.
- SW32Ti rotor (Beckman Coulter, Indianapolis, IN, USA) or equivalent.
- Beckman Coulter L-100XP Ultracentrifuge (Beckman Coulter, Indianapolis, IN, USA) or equivalent.
- Sorvall RC 6 superspeed centrifuge (Thermo Fisher Scientifi c, Grand Island, NY, USA) or equivalent.
- Real-time PCR system: Applied biosystems 7500 (Thermo Fisher Scientifi c, Grand Island, NY, USA) or equivalent.
- Lenti-X-qRT-PCR kit (Clonetech, Mountain View, CA, USA).
- Standard tissue culture equipment.
How to prepare the reagents?
- Prepare complete HEK-293 T media (DMEM with 10 % FBS and 1 % Pen/strep). Add 100 mL of FBS and 10 mL of Pen/ strep to 890 mL of DMEM ( see Note 5).
- Prepare PEI transfectionreagent. Heat 200 mL of dH 2O to 80 °C in a beaker on a stirring hotplate. Add 80.75 mg of PEI to the beaker and stir with a stir bar. Stir at 80 °C until the PEI is completely dissolved. Adjust pH to 8.0 with hydrochloric acid. Adjust to 250 mL with dH 2O. Filter sterilize with a 0.22 μm fi lter unit ( see Note 6).
- Prepare the required amount of plasmid DNA(for each respective plasmid) using a standard plasmid DNA preparation method. DNA must be endotoxin free.
Viral transfection and purification: how to do it?
- Seed the T175 fl ask with HEK-293 T cells. Grow HEK-293 T cells in a T75 starter fl ask prior to seeding the T175. Once the T75 starter fl ask is at 90–95 % confl uency aspirate media from the fl ask and gently rinse with sterile PBS. Incubate cells with 4 mL of dissociation reagent (e.g., TrypLE Express) at 37 °C for 5 min. Add 6 mL of media containing serum to deactivate the dissociation reagent, and triturate with 25–30 full strokes of a serological pipette to create a single cell suspension.
- Determine cell number per mL using a hemocytometer.
- Calculate the amount of the single cell suspension needed to seed the T175 fl ask with 3 × 10 7 HEK-293 T cells. Add cells to fl ask and bring the total volume of the fl ask to 28 mL with warm (37 °C), complete HEK-293 T media.
- Incubate T175 Flask at 37 °C with 5 % CO 2 overnight or until 80–90 % confl uency is reached.
- Prepare transfectionmixture. To create the complete transfection mixture, two separate solutions are fi rst prepared, a DNA solution and a PEI solution.
- Prepare the DNAsolution. In a 50 mL conical add 21.3 μg pNHP, 8 μg pHEF-VSVG, 7.2 × 10 11 copies of the pFIN transfer vector ( see Note 7), and 148.75 μL of 1.5 M NaCl. Bring total volume of DNA solution to 1.44 mL with dH 2O and mix well.
- Prepare PEI solution. In a separate 50 mL conical add 460.59 μL PEI and 148.75 μL 1.5 M NaCl to 1.04 mL dH 2O ( see Note 8). Mix well.
- Prepare the complete transfectionsolution by adding the PEI solution dropwise to the DNAsolution. Vortex vigorously for 1–2 min ( see Note 9).
- Incubate the complete transfectionsolution at room temperature for 20 min. The transfection solution should turn cloudy.
- Remove all media from the 80–90 % confl uent T175 fl ask. Mix the complete transfectionsolution with 28 mL of complete HEK-293 T media in a graduated cylinder. Gently add the transfection-media mixture to the T175 fl ask and be sure all cells are covered.
- Incubate the T175 fl ask at 37 °C and 5 % CO 2.
- Collect the supernatant containing virus( see Note 10). The supernatant is collected at 24 h post- transfectionand again at 40 h post-transfection. At the 24-h time point, collect the viral media in a 50 mL polypropylene conical. Replace the media with 28 mL of warm (37 °C) complete HEK-293 T media.
- Spin the collected viral media at 783 × g in the RC 6 (or equivalent) centrifuge for 5 min to pellet cell debris. Collect the supernatant and store in a fresh tube at 4 °C.
- At the 40 h time point, again collect the media and remove debris by centrifugation as in steps 12 and 13.
- Pool all viral media from the 24- and 40-h time points. Filter the viral media through the 0.45 μm Stericup-HV fi lter.
- Split the viral media into two thinwall polyallomer ultracentrifuge tubes ( see Notes 11 and 12).
- Carefully load the fi lled ultracentrifugation tubes into the SW32Ti rotor and spin at 80,000 × g for 2 h at 4 °C.
- Remove the supernatant from the viral pellet by carefully aspirating the media from the ultracentrifugation tube ( see Note 13).
- After removing the supernatant resuspend the viral pellet by gently overlaying the pellet with 10 μL of sterile DPBS in the conical ( see Note 14).
- Seal the conical tube with parafi lm and store at 4 °C overnight.
- After overnight incubation, place the sealed conical on ice. Gently shake the conical on an orbital rotating shaker for 2 h.
- Aliquot the resuspended virusinto working aliquots and store at −80 °C ( see Notes 15).
- Utilize one of your aliquots from step 22 and titer your viral prep using the Lenti-X titering kit following the manufacturer’s instructions.
- The current protocol utilizes the second-generation LV packaging system in which all necessary genes are separated into three distinct plasmids (the transfer vector, envelope plasmid and packaging plasmid). A third-generation packaging system is also available, in which the necessary genes are further separated into four distinct plasmids (transfer vector, envelope, and two packaging plasmids). Although the third generation system offers increased biosafety, it is also more cumbersome due to the increased number of plasmids. However, the current protocol is amenable to third generation packaging systems.
- The pNHP packaging helper plasmid encodes the following genes: Gag (encodes the capsid , nucleo capsid and matrix structural proteins), Pol (encodes reverse transcriptase, protease and integrase proteins), Tat (encodes protein for transactivation of transcription from viral LTR), Rev (encodes protein that mediates export of viral RNAs from nucleus), as well as vpr (mediates cell cycle arrest and assists in nuclear import) and vpu (enhances virion production and degradation of CD4 receptor in the host cell).
- pHEF-VSVG encodes the envelope glycoproteins derived from the VSV. LV vectors pseudotyped with VSV-Gexhibit a broad host cell range; however, the tropism, titers, and expression of levels LV vectors can be altered by pseudotyping vectors with glycoproteins from alternative enveloped viruses.
- The pFIN LV transfer vector contains a green fluorescent protein (GFP) gene followed by the woodchuck post transcriptional response element driven by the EF1α promoter. This expression cassette is flanked by HIV-1 LTRs. The pFIN vector also contains components from the LV genome absolutely necessary for viral packaging. If desired, the promoter and transgene can be easily replaced with a promoter or transgene of interest using standard cloning methods.
- All work should be done in a culture hood using sterile technique.
- Very little HCl is required to bring the PEI solution to a pH of 8.0. If too much HCl is added, use NaOH to bring the pH back to 8.0. Aliquot the PEI in 12 mL aliquots and store at −80 °C for future use.
- The size (in nucleotide bases) of the genetic material to be packaged in the lentivirus vector will alter the size of the pFIN plasmid and the corresponding weight of DNA needed for transfection . To maintain accuracy, determine the molecular weight of the pFIN plasmid containing your genetic material of interest and then calculate the total amount (in nanograms) necessary to transfect 7.2 × 10 11 copies of the pFIN plasmid. This can be done easily using an online DNA molecular weight calculator.
- PEI must be completely homogenous prior to use. If using a previously frozen aliquot, completely thaw at room temperature. Once thawed, heat solution to 55 °C to ensure all PEI is in solution. Let cool to room temperature before use.
- Adding the PEI dropwise to the DNA mixture prevents the rapid precipitation of DNA and ensures a homogenous DNA/ PEI solution. It is helpful to place the DNA mix on a stir plate and gently stir the DNA mixture while adding the PEI mixture dropwise.
- The quality of the transfection can be determined by viewing the cells under a fluorescent microscope. Alternatively if the GFP transgene was removed, transfection efficiency can be determined by observing the VSV-G mediated fusion of HEK293 T cells into multinucleated cell syncytia.
- If making larger preparations of lentivirus (using more fl asks or larger fl asks) it is necessary to concentrate the viral media prior to ultracentrifugation. This can be done using a column concentrator and centrifugation (e.g., Centricon-70 ultra-centrifugation column ) or using tangential fl ow filtration.
- Be sure to fi ll the ultracentrifugation tubes to the required level according to the manufacturer’s instructions or the tube will collapse during centrifugation.
- To remove the final few mL of media from the tube, tilt the tube at a 45° angle and continue to aspirate the media as it flows down the side of the tube away from the pellet. Do not disturb the pellet at the bottom of the tube.
- Due to the inherently “sticky” nature of the viral envelope and exposed glycoproteins, it is necessary to use siliconized pipette tips and tubes when handling the virus . For a detailed protocol on siliconizing.
- LV cannot be refrozen after thawing; as such it is best practice to aliquot the virus into small working aliquots.