Long DNA oligos containing the target sequence are cloned into LentiLox 3.7 (see
protocol). Once clones have been isolated, virus is produced by transfecting 293
cells and collecting supernatant. This supernatant is then used to infect cells
of interest directly, or concentrated for use in embryo infections.
LentiLox 3.7 (see sequence and map) is a lentiviral vector designed for inducing RNA interference in a
wide range of cell types, tissues and organisms. We have use this vector to
infect and efficiently silence proteins in hematopoietic stem cells and their
progeny, and have used infected embryonic stem cells and single cell embryos to
create transgenic animals (Rubinson and Dillon et al, Nature Genetics, 2003). These lentiviruses are pseudo-coated with VSV-G and are capable of infecting human
cells, and thus present important biosafety issues. This is explained here.
We use the 3rd generation packaging systems for lentiviral production originally
published by Dull T et al (J Virol. 1998 Nov;72(11):8463-71.). These vectors include: pMDLg/pRRE (gag/pol elements), pRSV-REV, and
pMD.G (env elements).
Sources of these packaging vectors include:
Invitrogen- Viral Power Lentiviral Support Kit K4970-00
Trono Laboratory, University of Geneva Medical School, Geneva, Switzerland
Naldini Laboratory, IRCC, University of Torino, Italy
Verma Laboratory, Salk Institute, San Diego, CA
- Plate 12 x 106 293.T in 20 ml on a 15 cm2
plate 24 hours before transfection. In general, two 15cm plates per
virus. It is essential that
the cells be well-maintained and of relatively low passage number.
- Mix the following
DNAs (made w/ Endo-free Qiagen Kits) in a FACS tube. The DNAs should be in Endo-free TE at a concentration of
For 3 plasmid system:
20 mg vector,
10 mg VSVG
15 mg D
For 4 plasmid, system (recommended),
10 mg VSVG
10 mg RSV-REV
10 mg pMDL g/p RRE
- Add 400 ml 1.25 M CaCl2 and 1.5 ml
H20 and mix by tapping gently.
The following steps are
done 1 plate at a time.
- Add 2 ml of 2X
HBS dropwise to DNA mixture while bubbling
with a Pasteur pipette.
When finished, continue to bubble for 12-15 seconds.
- Take plate of
293T out of the incubator (plate remains in incubator for long as
possible), and add transfection mixture dropwise all over the plate. Gently swirl plate from front to
back, and return immediately to incubator.
- 3.5 to 4 hours
later, remove media, wash 2x with 10ml warm
PBS, and add 20 ml warm D10 onto plate and place in incubator.
- 36-48 hours after
transfection, harvest viral supernatant and spin at 2000 rpm, 7 min at
4°C in a 50ml tube.
- Filter viral SN
through .45 um filter. Add
35ml of filtered supernatant to an ultracentrifuge tube. Balance tubes
with additional media. Cover tubes with small piece of parafilm.
(It is useful to titer some of the leftover supernatant to
determine if there is loss of virus during concentration.)
- Spin tubes using
a SW-28 rotor at 25,000 rpm, 90 min, 4°C. Decant liquid and leave tube
upside down on kimwipe for 10 min. Aspirate remaining media being
careful not to touch bottom of tube.
- Add 15ml cold PBS (for embryo infections,
or any volume you wish) and leave tube at 4°C O/N with no shaking.
- To resuspend, hold tube at angle and pipet fluid over pellet 20 times, being careful not
to touch pellet with tip.
It is expected that the pellet not be resuspended after this is complete. This pellet does
not contain virus and can be discarded.
- Aliquot or use
virus. Virus should be
aliquoted, flash-frozen in liquid nitrogen and
stored at -80. There should
be no change in titer with freezing concentrated virus. Avoid multiple
4x105 293.T cells/well in a 6-well plate 12-24 hours prior to
titering. It is helpful to have an additional
well as a negative control that you mock infect with D10+polybrene but
Make a stock solution of D10 with
Generate a 10-fold dilution series
of virus in the D10+polybrene.
Using 1.5mls/well you should have 1ml, .1, .01, .001, .0001, and
Incubate at 37
degrees O/N. Replace
media with fresh D10.
least 48 hours after infection trypsinize cells
for FACS analysis. (Trypsinize, inactivate with
media, spin, and resuspend in cold
analyze for EGFP expression and record the
percentage of cells that are EGFP positive.
a well that has between .1% and 10% of cells expressing EGFP to determine
calculation assuming 1% infection from the well with .01ml of virus.
.01 (percentage of cells that are EGFP
positive) x 4 x 105 = 4 x 103 positive
4 x 103 x 100 (dilution
factor) = 4 x 105 viral particles/ml
In general you should have at least 5 x 105 viral particles/ml for embryo