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The Retro-X™ Universal Packaging System and Retro-X™ Concentrator from Clontech has been validated for high titer virus production with Platinum Select MLP Retroviral shRNA-mir constructs (MLP). The system provides the GP2-293 packaging cell line optimized for retroviral production and all four commonly used envelopes including VSV-G, eco, ampho and 10A1. The choice of envelopes provides the broadest range of cell types allowing allow you to choose the tropism that is most appropriate for your RNAi experiment. Combined with the Retro-X™ concentrator these tools enable easy shRNA-mir delivery with quick access to high titer virus.
Schematic for retroviral packaging using Retro-X Universal Packaging System. The system consists of the GP2-293 Packaging Cell Line, which expresses viral Gag and Pol proteins, and a set of 4 viral env expression vectors. Cotransfecting an env vector with your retroviral construct allows you to select the ideal tropism for your virus. Package virus in 48 hoursHow the Universal Packaging System WorksThe GP2-293 cell line included in the Retro-X Universal Packaging System contains the MMLV gag and pol genes stably integrated in its genome. These two proteins are necessary for viral replication and particle formation, and their stable expression in the GP2-293 cell line contributes to high viral titers (Figure 1). The remaining component of the packaging process, a viral envelope gene, is supplied transiently and expressed to high levels when it is cotransfected with a user-supplied retroviral expression vector bearing the gene of interest. The co-transfected env gene encodes the viral envelope protein (Env) that determines the host range (tropism) of the virus, while high levels of Env help support the production of high titer supernatants. Schematic showing simple concentration protocol. Add Retro-X Concentrator reagent to clarified viral supernatant, incubate overnight at 4°C, and spin. Concentrate MLP shRNA-mir expressing viral particles without ultracentrifugationRetro-X Concentrator provides a fast, simple, and highly efficient method for concentrating MLP retroviral shRNA-mir viral particles. Just mix the retroviral supernatant with the Retro-X concentrator, incubate for a short period, and spin the mixture in a standard centrifuge.
Validated Protocols for MLP Packaging and ConcentrationObtain consistent high titers of MLP retroviral particles with the Retro-X Universal Packaging System. MLP vectors expressing shRNA-mir against a non-target control or K-ras (three different shRNA-mir) were packaged using the Retro-X Universal Packaging System and VSV-G, ecotropic, or amphotropic envelopes. Titers of retroviral supernatants were measured using flow cytometry of transduced HT1080 cells (VSV-G and ampho tropic virions) or NIH3T3 cells (ecotropic virions). All four MLP constructs produced consistent high titers. Packaging the MLP retroviral vectorThe MLP retroviral shRNA-mir vector has been validated for production of high titer retroviral particles using the Retro-X Universal Packaging System (Cat#631530). Virus production was consistent with the expected titers for each pseudotype, providing the broadest tropism for shRNA-mir delivery (Figure 3). The VSV-G envelope provided the highest titers, at 3–6 x106 IFU/ml while the ecotropic and amphotropic pseudotypes produced titers in the range of 1–4 x105 IFU/ml and 3–6 x105 IFU/ml, respectively.Effective concentration of MLP viral supernatants using Retro-X Concentrator. Retroviral supernatants were generated for the MLP-kRas-50 shRNA construct using the Retro-X Universal Packaging System. The viral particles were pseudotyped with 3 envelopes and titers were measured using flow cytometry of transduced HT1080 cells (VSV-G and amphotropic virions) or NIH3T3 cells (ecotropic virions). Each supernatant was then concentrated from 9 ml to 0.45 ml and re-titered. Concentrating MLP viral supernatants using the Retro-X concentratorThe Retro-X Concentrator was validated to concentrate MLP retroviral supernatants. 12-14X concentration of MLP viral supernatents were seen using the simple mix and spin protocol without the need for ultracentrifugation. More concentrated virus (up to a 100-fold increase) can also be obtained by reducing the volume of the resuspension step. Using the lower resuspension volume produced a titer of 1.2 x 108 (data not shown).Broad tropism
For example, ecotropic virus requires a specific receptor found only on mouse and rat cells, but pantropic viral particles pseudo-typed with the envelope glycoprotein from the vesicular stomatitis virus (VSV-G) transduce a large range of cell types from human to amphibians and nematode cells. MLP packaging has been validated with pantropic (VSV-G), ecotropic and amphotropic evelopes and was shown to transduce human and mouse cells. Retroviral-Gene-Transfer-and-Expression-User-Manual.pdf (1,166,172kb) Retro-X-Concentrator-Protocol.pdf (53,671kb) Retro-X-Universal-Packaging-System-Flyer.pdf (334,656kb) Retro-X-Concentrator-Flyer.pdf (191,289kb) pVSV-G-vector-information.pdf (85,523kb) p10A1-vector-information.pdf (86,111kb) pEco-vector-information.pdf (86,522kb) pAmpho-vector-information.pdf (89,688kb) GP2-293-Packaging-Cell-Line-COA.pdf (80,564kb) |
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