Start your RNAi experiments today! Let the MISSION team clone your shRNA into your vector of choice and then provide you with ready to use lentiviral particles.
- Select by methods other than puromycin, including G418 and FACS
- Confirm your lentiviral delivery through a fluorescent reporter
- Customize your shRNA sequence
- Eliminate tedious cloning and viral production
There may be times when classic drug selection for stable or semi-stable cell lines is not possible or desired. In addition, orthogonal methods of selection from puromycin may be required for the expression of multiple shRNAs or expression of shRNA concomitant with transgene expression.
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Selection by Fluorescent Proteins
Selection by G418
Alternatives to CMV Promoter
Vector Map and QC Information
Order Custom shRNA
Selection by Fluorescent Proteins
Sigma® now offers vectors containing multiple fluorescent protein alternatives (TurboGFP, TagCFP, TagYFP, TagRFP, and TagFP635). With fluorescent proteins in the vector, FACS and fluorescence microscopy techniques allow for rapid estimation of transduction efficiency and automated, rapid selection of cell populations that stably express the shRNA. The TurboGFP and far red fluorescent protein (TagFP635) are available in multiple backbones. Selection by fluorescent protein expression provides an orthogonal method when cells already stably express puromycin resistance. Sigma's MISSION bio-production team will customize the vector with your favorite shRNA sequence. To order either: contact your local sales representative, fill out our online request form, or email us at MISSIONRNAi@sial.com.
Selection by Neomycin
For selection by a second class of antibiotics, we offer vectors containing the neomycin resistance gene. These two vectors are pLKO.1-Neo and pLKO.1-CMV-Neo. Stable shRNA integrants can be found by selecting in G418 (A1720). As with puromycin, a cytotoxicity profile for your particular cell line will need to be generated. As an example see the puromycin protocol, but use G418 instead of puromycin. As a reference, a final concentration of 1 mg/ml of G418 has been used for selection of shRNA constructs in A549 cells. To order either: contact your local sales representative, fill out our online request form, or email us at MISSIONRNAi@sial.com.
Alternatives to CMV Promoter
Each type of promoter for your selection markers will offer different expression levels with different stabilities in each cell line. Importantly, the CMV promoter, while yielding strong expression in many cell lines, may not function in certain cells. Customer feedback and internal Sigma research and development work indicates that the CMV promoter will not function well in leukocytes, and certain mouse cells. In addition, the literature indicates that these promoters do not function in stem cells, a critical flaw for many researchers.1,2 We currently offer vector backbones that use other promoters than CMV. In fact, the vector backbone for the entire TRC collection utilizes the hPGK promoter for the puromycin selection and the U6 promoter for the shRNA. For data and protocols of how to utilize this vector in hES cell and lymphocytes, visit our shRNA protocols page. In addition, we offer the pLKO.1-Neo vector that uses the hPGK promoter driving the neomycin resistance gene for selection by G418. For certain mouse cells, the ubiquitin promoter (UbC) can be a viable alternative. The pLKO.1-puro UbC-TurboGFP construct and the pLKO.1-puro UbC-TagFP635 were generated for these types of applications. To order either: contact your local sales representative, fill out our online request form, or email us at MISSIONRNAi@sial.com.
Vector Map and QC Information
For vector sequence and maps of the custom cloning vectors visit our vector maps page.
The service includes cloning, sequencing, DNA preparation (500 ng) and lentiviral preparation (200 µl at 1 x 106 TU/ml, minimum titer via p24 assay). The order is typically shipped 4–6 weeks from the time the order is placed. To order either: contact your local sales representative, fill out our online request form, or email us at MISSIONRNAi@sial.com.
References
- Tang, F. C. et al. Stable suppression of gene expression in murine embryonic stem cells by RNAi directed from DNA vector-based short hairpin RNA. Stem Cells, 22, 93–9 (2004). Pubmed ID: 14688395
- Michibata, H. et al. Inhibition of mouse GPM6A expression leads to decreased differentiation of neurons derived from mouse embryonic stem cells. Stem Cells Dev., 17, 641–51 (2008). Pubmed ID: 18522499
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