Upon receipt of an order, bacterial cultures will be amplified from the glycerol stocks for use in purification of plasmid DNA. Viral particles will be produced from the plasmid DNA by transfection of ecotroic or amphotropic retroviral packaging cells or 293T cells + lentiviral packaging plasmids. The shRNA Shared Resource will then help the researcher with the infection of target cells. The resource can also select for individual or pooled puromycin resistant colonies, and after expansion, these cells will be provided back to the researcher.
The reagents could be used to study the effects of knockdown of individual genes or groups of genes in cultured cells, in vivo, or in transgenic mice after infection of ES cells. The genomic shRNA libraries can be used to screen for genes involved in various biological functions.
Other viral vector services:
Our resouce can also produce ecotropic, amphotropic, or polytropic retroviral supernatants from DNA constructs provided by researchers. A similar service is provided for lentivirus production. Costs for other transfection services and RNAi resources will be determined in consultation with shRNA Core personnel.
For best results, culture cells in the absence of antibiotics for several days in order to maximize the strength of the signal that is observed in PCR. Test supernatants to be used in PCR should be derived from cells that are at or near confluence. Each mycoplasma test requires ~250ul of cell culture supernatant. Supernatant may be heat-inactivated at 95°c for 5 minutes then frozen at -20°c until tested. Fresh culture supernatant may also be supplied for testing.