Gateway™ pEF-DEST51 Vector - FAQs

What are the benefits of the BLOCK-iT miR RNAi expression system over similar systems?

The BLOCK-iT miR RNAi expression system allows you to take advantage of promoter flexibility by choosing from a variety of Pol II promoters like CMV, Ubc, tissue specific, or inducible promoters. The miRNA vectors also allow you to clone multiple sequences in the same vector, thereby enabling you to target more than one gene or more than one location in a gene using a single plasmid. An additional advantage offered by some of the miRNA expression vectors is that transfection efficiency can be monitored with the EmGFP fusion partner.

Find additional tips, troubleshooting help, and resources within our RNAi Support Center.

What are the different promoter choices available with the BLOCK-iT miR RNAi expression system?

All the BLOCK-iT miR RNAi expression vectors are Gateway-adapted and contain the CMV promoter. If more specialized expression is required with a different promoter, the miRNA vectors allow for easy recombination with any other suitable destination vector that Thermo Fisher Scientific offers. A wide variety of Gateway Destination vectors that contain promoters such as EF-1apha or Ubc are available, and all be used for miRNA expression. MultiSite Gateway Technology vectors are also available which will enable you to use a tissue-specific promoter or a promoter of your choice to express miRNA.

Can I use E. coli containing an F' plasmid, such as Top 10F', to propagate a vector with the ccdB gene?

While the F' plasmid does contain the ccdA gene that can inhibit or reduce the toxicity of the ccdB gene product, the ccdA expression level is likely to be too low, or inhibition may not be complete, and the bacteria would still be exposed to the ccdB gene product and thus not grow. Therefore, bacterial strains containing the F' plasmid are not recommended as hosts for propagation of ccdB containing vectors.

For propagation of Gateway vectors containing ccdB, we recommend the One Shot ccdB Survival 2 T1R Competent Cells (A10460), which were specifically designed for that purpose. However, please note that these cells are not validated for propagation of other ccdB-containing vectors like the older pZErO plasmids, and in most cases they are not expected to work due to very high levels of ccdB protein expressed in those vectors.

Does the BGH poly A region in your vectors have a splice donor and splice acceptor (and therefore an intron)? What is the general function of the BGH polyA region?

There is no intron in the BGH polyA sequence. The BGH polyA signal (bases 1028-1252 in pcDNA3.1 vector) is the sequence that allows for polyadenylation of the RNA transcript. It is described in the following reference: The 3'-flanking sequence of the bovine growth hormone gene contains novel elements required for efficient and accurate polyadenylation. J Biol Chem.1992 Aug 15;267(23):16330-4

The reference states that the 3' untranslated region leading up to the poly adenylation core sequence (AATAAA) contains unique disperse non-consensus elements (consensus elements are poly U and GU-rich tracts) that serve to increase the efficiency of polyadenylation to a high degree, and that a 9-base sequence downstream of the core sequence facilitates efficient cleavage of the RNA strand. This leads to more stable and higher-abundance transcripts.