Section14:Appendix

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Appendix

shRNA-lentivirus system

shRNA can be delivered into cells either by transfection of plasmids expressing shRNA of the gene of interest or by infection of viral-packaged shRNA of the gene of interest in the form of lentiviral vectors. The following optimization of delivery of shRNA into cells is focused on the lentiviral shRNA vectors. The lentiviral library used here is created from a pLKO1 vector that carries a puromycin resistance gene and shRNA expression is driven from a human U6 promoter (Ref 11). The puromycin resistance gene has been used as a selection marker for infected cells harboring the shRNA vectors.

Cell based phenotypic RNAi assays using shRNA lentiviral vectors involves (1) viral production where shRNA vectors are packaged into lentivirus and (2) viral infection where the lentivirus harboring the shRNA vectors are transduced into the cells of interest.

Optimization of shRNA-lentivirus production

The production of shRNA-lentivirus involves the packaging of the shRNA vector into lentivirus and requires transfection of 2 plasmids which forms the packaging system, pCMVD8.9 (Ref 12, 17) and pHCMV-G (Ref 16). In the transfection process, the key factors to be optimized are the seeding density, transfection reagents used, concentration of plasmids and the ratio of transfection reagent to plasmids. For concentration of plasmids, the usual practice includes concentration ranging from 100ng to 200ng. The plasmid concentration to transfection reagent ratio to be tested usual includes 2:1, 3:1 and 3:2. Viral production can be performed using the protocol published by the Broad institute at http://www.broad.mit.edu/genome_bio/trc/publicProtocols.html.

GFP control vector is used for optimization purpose and can be viewed briefly under the microscope to assess fluorescence. The number of infectious units in the viral supernatant calculated as IU/ml is assessed by infecting cells with generally 2ml of virus and counting survival of cells after puromycin treatment. The viral titer determination is important to assess the amount of virus to be used in infection for cell based assay. An acceptable range for viral titer is 2 x 106 to 2 x 107. A variety of commercial kits (p24 ELISA) are now available to determine titer.

shRNA-lentivirus production protocol (96-well microplate format)

1. Dilute D8.9 to 9ng/ml, vsv-g to 1ng/ml and shRNA to 25ng/ml.
2. Add 6ul per well (150ng) of shRNA and 5ml each of D8.9 (45ng) and vsv-g (5ng) to the shRNA.
3. Dilute transfection reagent (e.g. Fugene 6 from Roche) in Opti-Mem to a volume of 14ml per well, that is, 0.6ml of reagent to 13.4ml of Opti-Mem. The final ratio of transfection reagent:DNA should be 3ml:1mg.
4. Add diluted transfection reagent (e.g. Fugene 6 from Roche) to the plasmid mix to a final volume of 30ml per well and incubate for 30-45 min at room temperature.
5. Transfer Fugene/DNA complex to HEK293T cells grown overnight seeded at 25000 cells per well in low antibiotic growth media. Incubate for 18h at 37°C.
6. Replace media with 170ml of high serum growth media and incubate for further 24h at 37°C.
7. Harvest 150ml of viral supernatant and add 170ml of high serum growth media and incubate for another 24h at 37°C.
8. Harvest another 150ml of viral supernatant and discard cells.
9. Pool viral supernatant and use for infection.

Examples of plate layout for control plates to quality control RNAi synthetic lethality screens.