Lentiviral vectors are ideally suited vehicles for just a wide range of research applications. Based on pseudotype, lentiviruses infect a variety of cell types, non-discriminately transducing both dividing and non-dividing cells. As opposed to other popular vector delivery systems, lentivirus stably and rapidly integrates genetic payload in the host genome enabling long-term studies in vivo. Current lentiviral vector systems can accommodate upwards of ten kilobases of foreign DNA , although promoter and enhancer elements slow up the practical height and width of gene open-reading frames to perhaps 5-6 kilobases, which is sufficient to accommodate most genes commonly studied within the mammalian genome. Genetic integrants also adopt native chromatin conformation ideal for easy use in gene regulation studies  and particular lentiviral pseudotypes are actually exploited for selective infectivity whereby cells interesting are preferentially transduced; as an example, VSV-G pseudotype displays broad tropism but preferentially targets neurons within the nerve fibres. Aside from the ability to transduce virtually all immortal cell lines with high efficiency, lentivirus has been used successfully in the growth and development of transgenic animals and vivo types of disease (reviewed in ), and involvement in gene therapy applications is constantly on the increase.
Research laboratories can produce current-generation lentiviral vectors in approved biosafety level-two containment areas with the necessity for specialized equipment. For optimal safety consideration, the next generation of lentivirus affords the very best level of protection, whereby minimal genetic elements are split among four plasmids that need to be expressing simultaneously in individual cells for successful viral production [4-6]. After transfection and subsequent expression with the proteins encoded because of the transiently transfected plasmids, the cell medium supernatant contains active lentiviral particles immediately usable to transduce other cells of great interest. However, investigators routinely report problems in obtaining sufficient levels of lentivirus for particular projects, and lentivirus used in vivo has experienced varied reports of success within the literature. Difficulties with low titer and toxicity in purified preparations plague common entry to lentiviral vectors. Traditional methods for manufacture of lentiviral titers utilize calcium phosphate precipitation to transfect some required plasmids in to a packaging cell line, mostly HEK-293T cells . Within our experience, this standard approach leads to high variability in transfection efficiencies and corresponding titers, and also the concentrated virus can be highly toxic to cells in downstream experiments.
Here, we describe many advances inside the production of lentiviral vectors. There are numerous new releases now commercially accessible which have clear benefit over predecessor equivalents. Using these protocols and associated products, we have virtually eliminated toxicity regarding lentiviral preparations and routinely obtain titers inside mid to high 1010 transduction units (TU) per milliliter, and, more importantly, obtain consistency in production.
The third-generation lentivirus system employs several enhanced safety measures over the second-generation packaging approach. Three packaging plasmids allow expression in trans of proteins required to produce functional virus. The packaging plasmids pLP1, pLP2 and pLP/VSVG (Invitrogen) were modified to prevent stretches of homology within the plasmids to prevent recombination as well as contain optimized promoter and enhancer elements for expression in HEK-293FT cells. These packaging plasmids are compatible with a number of lentiviral expression plasmids. To the experiments described here, a lentiviral vector encoding eGFP (cFUGW), WPRE, and cPPT was utilized. Preference ought to be directed at lentiviral expression plasmids containing the cPPT and WPRE elements that considerably increase transduction efficiency, specifically in primary cells.
Packaging cell line
A serious limitation and supply of variability in virus production stems from variations inherent in commercially accessible serum products, where lot-to-lot variations of 10,000-fold are already described for a few growth factors. We sought to remove the employment of serum products during virus production and identified a commercially accessible cell line (HEK 293-FT cells, Invitrogen) amenable to serum-free media formulations. 293-FT cells are clonal derivatives on the fast-growing 293-F cell line variant and stably express the SV40 large T antigen driven on the pCMVSPORT6TAg.neo construct. Over-expression from the large T antigen allows enhanced episomal replication of packaging and lentiviral expression plasmids containing the SV-40 origin of replication and usually enhances lentiviral production through additional unknown mechanisms. 293-FT cells were switched from the manufacturer´s recommended medium with a low/no serum-compatible medium (Opti-MEM, Invitrogen) supplemented with 5% fetal-bovine serum (FBS) and 500 µg/ml G418 for growth and maintenance. Culture vessels are maintained at 37°C at 5% CO2 in 95% RH.
Lentivirus supernatant production
HEK-293FT cells maintained in Opti-MEM (with Glutamax) supplemented with 5% FBS were plated to approximate 50% confluence in Nalge Nunc Nunclon T-175 flasks coated with polylysine (flasks were helped by 0.1 mg/ml polylysine for 60 minutes and washed 3 x with mineral water immediately previous to use). Sixteen hours later, the medium was substituted for 20 ml of serum-free Opti-MEM supplemented with 25 µM chloroquine. 10.5 µg LP1, 7 µg LP2, 10.5 µg pVSV-G, and 9 µg of lentiviral vector were included with 2 ml of serum-free Opti-MEM. 100 µl of FuGENE® HD Transfection Reagent was added as well as the mixture was briefly vortexed and incubated at room temperature for quarter-hour. This mixture was added straight to the HEK-293FT cell layer while the flask was gently agitated to distribute the transfection complexes. Eight hours later, 10 µM sodium butyrate was added right to the flask medium; 1 day following the addition in the transfection complex, the medium was discarded and replaced with 20 ml of serum-free Opti-MEM without any supplementation. Another twenty four hours later, the medium was collected in a 50-ml tube and another 20 ml of Opti-MEM was included with the T-175 flasks. Medium supernatant was centrifuged at 3,000 x g for ten mins and decanted into another 50 ml tube and held at 4˚C. After 1 day, a second collection was included with the 1st collection and centrifuged again at 3,000 x g for 10-20 minutes. The supernatant was filtered by way of a 44-µm membrane. Virus is aliquoted and stored at -20˚C. Thus, each T-175 flask yields 40 ml of viral supernatant that will yield 1-5 x 107 infective particles per milliliter. The protocol can be adjusted to support larger preparations by appropriate scaling.
For lentiviral applications over and above cell line use, concentration is generally important to achieve high transduction rates. Moreover, concentration is needed for titer estimation using p24 protein measurements. To concentrate the viral supernatant, 100 µl of Opti-prep density gradient medium (Sigma) is added to Beckman Ultraclear centrifuge tubes, and ~38 ml of supernatant is added per tube. The density gradient medium prevents harsh pelleting and resuspension steps that otherwise diminish titer. The supernatant is centrifuged using a swinging bucket rotor for three hours at 50,000 x g plus the top 37 ml are manually removed having a pipette. The end 1 ml boasts a 40 x viral preparation helpful for ex vivo applications that has a usual titer up to ~5 x 108 TU per ml.
For in vivo applications, 1-ml 40 x aliquots are combined into sterile microcentrifuge tubes and centrifuged for 18 hours at 20,000 x g at 4˚C. The supernatant is carefully removed along with the pellet is resuspended by gentle manual pipetting on the limits of solubility using pre-warmed serum-free Opti-MEM. Viral pellets may also be dissolved in pre-warmed PBS, although Opti-MEM contains small quantities of protein that act surfactants to further improve virus solubility. Virus is aliquoted into 5-µl fractions and stored at -80˚C, through an expected titer of ~5 x 1010 TU per ml.
HEK-293FT cells or other cells of interest are plated with low confluency onto polylysine-coated 96-well plates with 100 µl serum-free Opti-MEM per well. The very next day, lentivirus preparation is included in the primary well (recommended 50 µl for 1 x supernatant, 10 µl for 40 x concentrate, and a couple of µl for high concentration virus) and volume is delivered to 200 µl with additional Opti-MEM. After mixing, 100 µl are taken out of the very first well and included in another well, mixed, and 100 µl are stripped away from that well, combined with the following etcetera, thereby accomplishing single:2 dilution number of virus. If your lentivirus encodes a fluorescent protein, a rightly containing an easily countable volume of fluorescent cells is identified: each infected cell represents a transducing unit. If the lentivirus isn’t going to encode a fluorescent protein, cells are fixed and immunofluorochemistry is used to distinguish transduced cells.
Results and Discussion
The primary measures in optimizing production of lentivirus involved rectifying steps of possible variation and excretion of components that can cause downstream toxicity. We identified a cell line (HEK-293FT cells) that’s amenable to low or no serum formulations that produce the best stages of lentivirus. Besides Opti-MEM, which contains animal-derived components, we used two other medium formulations which are chemically defined, namely CD-293 medium and Freestyle-293 media (Invitrogen). We experienced a 20% reduction of lentivirus production when substituting to CD-293 medium, and an 80% decrease in lentivirus production with Freestyle-293 medium (data not shown).
Traditional calcium phosphate transfection produces toxicity in cells, creating cell-lifting problems, and necessitating additional medium changes. Cell lifting and death during viral production releases cell components that become co-concentrated with viral particles, thereby introducing toxic components to viral preparations. We found that FuGENE® HD Transfection Reagent co-transfects the 4 plasmids needed to produce virus with 100% efficiency into low-serum modified HEK-293FT cells. FuGENE® HD Transfection Reagent, as opposed to calcium phosphate, won’t cause cell lifting and death, plus doesn’t need an intermittent medium change.
We sought to help expand maximize viral production and transfection efficiency of cells transfected using FuGENE® HD Transfection Reagent through pre-management of cell lines with chloroquine and further treatment with sodium butyrate (Figure 1). Chloroquine is shown to lessen the degradation of plasmid-containing transfection complexes through partial neutralization on the pH within lysosomal compartments . We found that chloroquine gives both immediate and long-term help to viral production, probably by helping the effective concentration of active plasmid DNA in cells. Sodium butyrate, which exerts a broad-spectrum effect on transcriptional activity, likewise increases viral production presumably through a combination of up-regulating viral promoters and boosting protein production output capability . The amalgamation of chloroquine and sodium butyrate treatment modestly increases viral output compared with either treatment alone, perhaps caused by a maximal viral output limitation of transfected cells.
In vivo entry to lentivirus demands particularly pure and concentrated preparations. To determine whether or not the protocol described herein produced virus from the required caliber, we injected a couple-µl number of 4 x 1010 TU virus encoding eGFP in to the anterior region of the mouse button caudate putamen (Figure 2). After 7 days, we analyzed brain sections through immunohistochemistry (Figure 2, panel a) and immunofluorescence (Figure 2, panel b). Broad infectivity with minimal toxicity throughout the needle site and with the striatum demonstrates the premium quality of lentiviral preparations produced using FuGENE® HD Transfection Reagent.