Biology Reference
In-Depth Information
systems can suppress the expression of genes of interest in the
transduced cells, but the shRNAs are driven by an RNA polymerase
III promoter, whereas the miRNAs are transcribed by a polymerase
II promoter. A useful tool for designing gene silencing vectors
based on the first technology is the silencing lentiviral library, made
by the RNAi consortium (
http://www.broadinstitute.org/rnai/
mouse genes. The carrying vector (pLKO.1, available through
Sigma-Aldrich and Open Biosystems) has an RNA polymerase III
U6 promoter, which drives the expression of the shRNA, and a
puromycin-resistant cassette that could allow selection of trans-
duced cells. The second technology (based on miRNA) has been
demonstrated to elicit a stable and specific knockdown in vitro and
in vivo [
31
] and using a tissue-specific polymerase II promoter can
have the significant advantage of allowing a cell and time specific
targeting of the gene expression that is not possible with the
shRNA system.
3
Materials
3.1 Cell Culture,
Transfection, and Viral
Vector Production
1. Dulbecco's Modified Eagle Medium with GlutaMax (DMEM
GlutaMax, Gibco)
2. Iscove's Modified Dulbecco Medium (IMDM, Sigma)
3. Fetal Bovine Serum (FBS, Euroclone)
4. 100× Glutamine: 200 mM glutamine (Gibco)
5. 100× Pen/Strept: 10,000U/ml Penicillin, 10,000 μg/ml
Streptomycin (Gibco)
6. Sodium butyrate (Sigma)
7. Dulbecco's Phosphate Saline Buffer (PBS)
8. Trypsin solution (Gibco): 0.05 % Trypsin, 0.53 mM ethylene-
diaminetetraacetic acid (EDTA)
9. Tris-EDTA (TE) buffer: 10 mM Tris pH 8.0 (Sigma), 1 mM
EDTA (Sigma)
10. Collagenase (Sigma)
11. 100 mm, 150 mm and 6 wells plates
12. 2× HBS: 281 mM NaCl (Sigma), 100 mM HEPES (Sigma),
1.5 mM Na
2
HPO
4
pH 7.09 (Sigma), filter sterilized and stored
at −20 °C
13. CaCl
2
solution: 2.5 M CaCl
2
(Sigma), filter (0.22 μm, MILLEX
GP Millipore) and store at −20 °C
14. Sterile water (SALF laboratories)
15. Syringe-driven filter units (0.22 μm)
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