An Optimized Protocol for Packaging Pseudotyped Integrase Defective Lentivirus

Integrase Mutagenesis and Reporter Construction

The pPACKH1-GAG plasmid (SBI, Mountain View, CA) contains the structural (gag), replication
(pol) and envelop (env) gene. The pol contains a functional domain of integrase, which
is responsible for provirus integration. As shown in Fig. 1a, we designed and synthesized primer pairs to carry out site-directed mutagenesis
using high fidelity DNA polymerase followed by DpnI (NEB, Ipswich, MA) treatment and
transformation. The primers used for D64A (forward 5?- aggaatatggcaactagcttgtacacatttagaag-3?;
reverse 5?-ctaaatgtgtacaagctagttgccatattcctggac), D116A (forward 5?- gtaaaaacaatacatacagccaatggcagcaatttcacc-3?;
reverse 5?- gaaattgctgccattggctgtatgtattgtttttactggc-3?), D152A (forward 5?-caaagtcaaggagtagtagcatctatgaataaagaattaaagaaaatt;
reverse 5?- ctttaattctttattcatagatgctactactccttgactttgggg-3?), and triplet mutation
in basic region (forward5?-gtgacataaaagtagtgccagcagcacatgcaaagatcattagggat-3?; reverse
primer 5?-atccctaatgatctttgcatgtgctgctggcactacttttatgtcac- 3?). Using different combinations
of primer pairs, we have generated five integrase mutants as confirmed by double stranded
DNA sequencing (SeqeneTech, Mountain View, California).

Cell Culture and Transduction

Human embryonic kidney HEK293-TN (LV900A-1, SBI, Mountain View, CA) and fibrosarcoma
HT1080 (ATCC, Manassas, VA) cells were maintained in high glucose Dulbecco’s Minimal
Essential Medium (DMEM) supplemented with 10 % FBS, 2 mMGlutaMax (Life Technologies),
100 U/ml penicillin and 100 U/ml streptomycin. Transduction reagent TransDux (LV850A-1,
SBI, Mountain View, CA) was used to infect cells with virus to enhance the transducing
efficiency. CD511B-1 (GFP reporter) and LV605A-1 (DualGFP-RFP reporter) from SBI were
the constructs used to make WT and integrase defective lentiviral particles for most
of the studies. These constructs were assembled by a combination of PCR and seamless
fusion method as reported before 15], 16].

STO Culture and Actinomycin Treatment

STO cells were cultured in DMEM high-glucose (Life Technologies), supplemented with
2 mMGlutaMax (Life Technologies), and 1 % nonessential amino acid (Life Technologies).
Cells were treated with 10 ?g/ml actinomycin C (Sigma) for 3 h. The actinomycin C-treated
STO non-dividing cells were extensively washed in PBS and plated at 35,?000 cells/cm
²
in gelatin-coated tissue culture dish.

Microscopy and Reporter Gene Assay

All microscopy was performed on live cells with a LEICA DMI3000B microscope following
48?~?72 h transduction. Data collection and processing were performed with LAS 3.8
software.

Lentiviral Titration

Pseudoviral titer was performed using a real time PCR based kit (LV961A-1, SBI, Mountain
View, CA). This kit measures the copy numbers of integrated gene after transduction.
Briefly, HT1080 cells were transduced in the presence of 1?×?TransDux reagent (LV850A-1,
SBI, Mountain View, CA) for 72 h. Subsequently cells were lysed and DNAs were isolated
according to manufacturer’s instructions. PCR reactions were performed with primers
for amplifying a conserved genomic DNA sequences or WPRE in the transgeneusing Applied
Biosystems 7300 Real time PCR System. PCR condition: 50 °C, 2 min; 95 °C, 10 min;
followed by 40 cycles of 95 °C, 15 s and 60 °C for 1 min, followed by a dissociation
step.

Standard Operating Procedure for Lentiviral Packaging

Reagents

The expression constructs CD511B-1 and LV605A-1 (SBI, Mountain View, CA) are packaged
into VSVG pseudotypedviral particles with proteins produced by the lentiviral packaging
plasmid mix (LV500A-1, SBI, Mountain view, CA). The lentiviralpackaging system consists
of an optimized mixture of three plasmids, including pPACKH1-GAG, pPACKH1-REV and
pVSVG. To produce integrative competent lentiviruse, the pPACKH1-GAG has a wild-type
pol gene. To produce non-integrative lentiviruses, the pPACKH1-GAG with a mutated
integrase (an integrase domain in pol gene) was used instead of the wild-type. Other
reagents include virus producer cell lineHEK293TN (LV900A-1, SBI, Mountain View),
transfection reagent PureFection (LV750A-1, SBI, Mountain View), and virus precipitation
solution (LV810A-1, SBI, Mountain View).

Additional reagents include DMEM high glucose with sodium pyruvate and l-glutamate
(Invitrogen, Cat. #111995073), Fetal bovine serum (Invitrogen, Cat. #16000036), Penicillin/Streptomycin
(Invitrogen, Cat. # 15070063), Trypsin-EDTA (Sigma, Cat. #T3924), hematopoietic plate
cell counter.

Step-by-step Protocol (bench time 110 min, total time 6 days)

Day 1

Seed 293 TN producer cells (30 min)

18?~?24 h prior to transfection, wash cells with pre-warmed DPBS once and add 2 mlTrypsin-EDTA
to the culture flask. Incubate for 5 min or until cells become detached from surface
and add 10 ml of complete medium to neutralize the digestion enzyme. Precipitate cells
by centrifugation at 300 rpm for 5 min at room temperature. Re-suspend cell pellet
in 5 ml complete medium and count cells. Seed 293TN cells at 7?~?8?×?10
⁶
/15 cm
²
in 20 ml complete culture medium with antibiotics, until the cell density reaches
70?~?80 % confluence for transfection.

Day 2

Transfecting 293TN Producer cells with lentivector expressing reporter (CD511, LV605)
and lenti viral packaging plasmid mix expressing WT and mutant integrase (bench time
30 min)

1.
Prepare lentivector DNA and packaging plasmid mix

Add 1?~?1.6 ml plain DMEM (serum and antibiotic free) to an Eppendorf tube; add 4.5 ?g
lentivector and 45 ?l packaging plasmid DNAinto the DMEM. Mix by pipetting for 10
times.

2.
Prepare transfection mix

Add 55 ?l PureFection into the same tube. Vertex for 10 s and incubate the DMEM-Plasmid-PureFectionmixture
at room temperature for 15 min.

3.
Apply transfection mix to producer cells

Add DMEM-Plasmid-PureFectionmixture drop-wise to the dish, and swirl to disperse evenly;
return the dish to the cell culture incubator at 37 °C with 5 % CO
₂
; change the medium 12?~?24 h after transfection (optional).

Day 4 and Day 5

Harvest packaged viruses (bench time 30 min; total 2 h)

48 h after transfection, collect the culture medium containing pseudoviral particles
into a 50-ml sterile, capped conical centrifuge tube and store at 4 °C until next
harvest. Re-fill producer cells with 10 ml fresh complete medium slowly, and return
dishes to the cell culture incubator.

72 h after transfection, collect the culture medium and combined it with the collection
at 48 h. Centrifuge at 3000?×?g for 15 min at room temperature to pellet cell debris.
Transfer the viral supernatant into a new tube and add 1 volume of cold PEG-it Virus
Precipitation Solution (4 °C) to every 4 volumes of Lentivector-containing supernatant.
Larger volume precipitation of lentiviral particles can be achieved by using the Corning
250 ml polypropylene centrifuge tube (Cat. #430776), following manufacturer’s instructions:
refrigerate overnight or for at least 12 h. Lentivirus-containing supernatants mixed
with PEG-it Virus Precipitate Solution (LV810A-1, SBI, Mountain View, CA) are stable
for up to 4?~?5 days at 4 °C.

Day 6

Collect and concentrate psuedotyped viral particles (bench time 20 min; total 1.2 h)

Centrifuge supernatant/PEG-it mixture at 3000?×?g for 30 min at 4°C, and transfer
supernatant to a fresh tub. After centrifugation, the lentivector particles will appear
as a beige or white pellet at the bottom of the vessel. Spin down residual PEG-it
solution by centrifugation at 3000?×?g for 5 min; remove all traces of fluid by aspiration,
and avoid disturbing the precipitated lentiviral particles in pellet. Re-suspend lentiviral
pellets from 1/10 to 1/100 of original volume using cold, sterile PBS or DMEM containing
25 mM HEPES buffer at 4 °C. Aliquot into cryogenic vials and store at ?70 °C.

Trouble-Shooting Advice

1. Low viral titer (105 ifu/mL) can be caused by a number of reasons. The most common
ones include:(1) poor transfection efficiency due to too high or too low density of
producer cells. (2) Poor quality of lentivector or packaging plasmid DNA. (3) Inadequate
ratios of plasmid mix to the transfection reagent. To avoid these, plate cells to
achieve 70?~?80 % confluency at transfection stage and use endotoxin-free plasmid
purification kit to prepare lentivector expressing the transgene and packaging plasmids.

2. If problem remains, other factors to be consideredare: (1) producer HEK293TN cells
are of poor quality due to over confluence; (2) cells may be contaminated by mycoplasma;
(3) too many passages (20) of cells. In those cases, one should revive a new batch
of producer cells and maintain them in the logarithmic growth phase.

3. Other factors which could affect virus quality are: The packaging limit for the
lentiviral system, which is ~8.5 kb from 5????3? LTR. The efficiency of packaging
drops significantly when cDNA size increases. For a 3 kb insert, the titers and expression
level can be 10-fold less compared to that ofa 1 kb insert. Repeated sequences can
also cause problems in transgene expression as recently reported 17].

Safety Guide to Packaging and Transduction of Target Cells

The use of HIV-based pseudotypedlentiviruses falls within NIH Biosafety Level 2 criteria
due to the potential biohazard risk of possible recombination with endogenous viral
sequences to form self-replicating virus, or the possibility of insertional mutagenesis.
For a description of laboratory biosafety level criteria, consult the Centers for
Disease Control Office of Health and Safety Web site. It is also important to check
with the health and safety guideline at your institution regarding the use of lentiviruses
and always follow standard microbiological practices.