Supplemental File 3
VIRUS TRANSDUCTION PROCEDURES
We set up our virus transduction procedure to minimize cytotoxic effects (Forte et al., 2007; Jori et
al., 2007; Jori et al., 2005; Jori et al., 2004; Napolitano et al., 2007).
siRNA design and synthesis
Hairpin siRNAs targeting human BRG1 mRNA were selected using the design algorithm developed
by Cenix Bioscience GmbH (Reynolds et al., 2004). The siRNAs target several regions of human
BRG1 mRNA (NCBI accession NN_003072.2).
The control siRNA targets the firefly (Photinus pyralis) luciferase gene (GenBank accession
number X65324) (Harborth et al., 2001). The control siRNA has no target mRNA in the human
transcriptome.
For each siRNA, two complementary hairpin siRNA template oligonucleotides were synthesized
(Eurofin MWG Operon, Ebersberg, Germany) as a 55-mer. They included the 19-mer stem with a
2-nucleotide overhang and the loop sequence 5’ TTCAAGAGA 3’. The 5’ ends of the two template
oligonucleotides form the Xho I and Spe I restriction site overhangs, which facilitate efficient
directional cloning into the Shuttle Vector (see below).
Adenoviral production
The recombinant adeno-siRNAs were produced using the pSilencer adeno 1.0-CMV System
(Applied Biosystems, Monza, MI, Italy) according to the manufacturer’s instructions. Briefly, the
two pairs of complementary 55-mer oligonucleotides related to the BRG1 and luciferase mRNAs
were annealed to form the siRNA template inserts. The siRNA template inserts were successively
ligated into the linear Shuttle Vector 1.0-CMV supplied with the kit. One Shot TOP10 chemically
competent E. coli cells (Invitrogen Italy, Milan, Italy) were transformed with the ligation products
and plated on medium containing ampicillin to select for transformants. Clones were screened for
the siRNA inserts by digestion with Xho I and Spe I (Roche Italia, Milan, Italy). Positive clones
were then expanded for large-scale plasmid preparation. The inserts were sequenced using the
primers provided by the kit to verify that that the hairpin siRNA templates had the correct
sequences.
Shuttle Vector 1.0-CMV containing the siRNA templates and the Adenoviral LacZ Backbone
provided with the kit were linearized by digestion with Pac I (Roche Italia, Milan, Italy). The two
plasmids were then transfected using calcium chloride into 293 primary embryonic kidney cells
(HEK-293), a packaging cell line that supplies the E1 function in trans. Homologous recombination
occurred in the region of overlap between the plasmids, thereby producing the recombinant adenosiRNAs (Ad-siRNAs).
HEK-293 cells were harvested 12 days after transfection, when at least 60% of the cells had become
infected and detached from the culture plate. To obtain enough of the viruses for subsequent MSC
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transduction, recombinant Ad-siRNAs were expanded by the infection of HEK-293 cells, purified
and titrated.
Virus purification
The AdEasy™ Virus Purification Kit (Stratagene, La Jolla, CA, USA) allows for the purification
and concentration of Adenovirus (from Ad5 strains) with Sartobind® syringe filters containing an
ion-exchange membrane adsorber that selectively binds adenoviral particles. Once bound, viral
particles can be further purified by washing away the nonspecifically bound protein before elution.
Briefly, infected HEK-293 cells were harvested when most cells showed full cytopathic effects and
detached from the culture flasks. Cells were centrifuged, and both the pellet and supernatant were
retained. Pellets were dissolved in 10 ml of supernatant, and cells were lysed by three freeze/thaw
cycles. Cells were then centrifuged to remove cellular debris and recombined with the reserved
supernatant. The prepared supernatant was passed slowly, drop by drop, through the Sartobind
columns. After a washing step, the virus was collected with elution buffer.
Virus titer determination by the end-point dilution assay
For the titer determination of each virus preparation, we plated HEK-293 cells in two 96-well plates
at ~70% density a day before the assay. We prepared serial dilutions of the virus preparation. In
each row of 96-well plates, we added the same viral dilution starting from 10–1. Cells were then
incubated for 10 days at 37°C.
At the end of the incubation period, we checked each well for cytopathic effects (CPE) using a
microscope. For each row, we counted the number of wells displaying CPE. A well was scored as
CPE-positive even if only a few cells showed cytopathic effects. We then calculated the fraction of
CPE-positive wells in each row. The viral titer was then determined as follows:
Titer (pfu/ml) = 10 1 + Z (X – 0.5).
Z = Log10 of the dilution (= 1 for a 10-fold dilution)
X = the sum of the fractions of CPE-positive wells.
Determination of the percentage of transduced cells
MSC cultures were transduced with 5, 10, 20, 50, 100, 250 or 500 MOIs of control Ad-siRNA. The
percentage of transduced cells was then calculated by detecting the β-galactosidase activity of the
LacZ reporter gene contained in the Adenoviral LacZ Backbone used for recombinant Ad-siRNA
production using an in situ detection β-gal staining kit (Roche Italia, Milan, Italy) according to the
manufacturer’s instructions.
Determination of the cytotoxic effect
For the cell death assay, 3 x 103 MSCs were seeded at equal densities in 96-well plates and
transduced with 5, 10, 20, 50, 100, 250 or 500 MOIs of control Ad-siRNA. Forty-eight hours after
transduction, cell death was determined by measuring the metabolic activity of cellular enzymes
released in the culture medium using the CytoTox 96 Cell Death Assay kit (Promega) according to
manufacturer’s instructions.
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Our experimental conditions reduced greatly the cytotoxic effects associated with virus transduction
experiments. See the table below.
MOI
% of dead cells
% of infected cells
0
5
20
50
100
250
500
3 ± 0.8 3 ± 0.9 7 ± 1.8 7 ± 2.1 12 ± 1.8 20 ± 2.7 24 ± 2.3
40 ± 8 80 ± 6 93 ± 5
95 ± 6
95 ± 12 94 ± 13
References
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RB2/P130 genes cooperate with extrinsic signals to promote differentiation of rat neural stem cells.
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