AdenoVator™

Q. Will extra bases in the 5' and 3' UTRs (Untranslated regions) of my gene cloned into a transfer vector affect protein expression?

A. For optimal protein expression, UTRs have to be as small as possible, especially in 5' to avoid formation of secondary structures in the mRNA. A minimal length (7-8 bases) in front of the ATG is acceptable, especially if the Kozak consensus sequence (GCCGCCACCATGG) is included. There are no special requirements for the 3'UTR.

Q. Why are there only 1-2 cloning sites after the CMV5 promoter in some of your transfer vectors?

A. The powerful CMV5 promoter available in some of our transfer vectors is optimal for the overexpression of proteins. In these vectors, only one or two cloning sites are available instead of a multiple cloning site (MCS) in order to achieve high expression levels. Because of this, the number of untranslated bases between the promoter and the start codon must be minimized and it is recommended to avoid adding 5' untranslated bases (i.e. an MCS) as much as possible when cloning in order to obtain optimal expression.

Q. What is the difference between the CMV and the CMV5 promoters?

A. The CMV5 is an enhanced version of the CMV promoter. The CMV5 promoter in our AdenoVator™ vectors was constructed by the insertion, downstream of the transcription start site of the human CMV IE promoter-enhancer, of the adenovirus tripartite leader (Ad-tpl) with the adenovirus major late enhancer bracketed by splice donor and acceptor sites. The Ad-tpl binds translation-initiating proteins much more efficiently than most messages. Viruses with CMV5 express about 6-10 fold more protein in 293 cells than viruses with CMV.

Reference:

Massie, B., F. Couture, et al. (1998). "Inducible overexpression of a toxic protein by an adenovirus vector with a tetracycline-regulatable expression cassette." J Virol 72(3): 2289-96. confounded

Q. Is it possible to first transform the BJ5183 with pAdenoVator E1/E3 and then make this recombinant strain electrocompetent for transformation with the linearized transfer vector?

A. Yes, it is possible. However, we do not recommend it since BJ5183 cells support the RecA recombination system. Our researchers have shown the occurrence of unwanted recombinations in laboratory tests. Therefore, we recommend growth and amplification of plasmids should not be performed in that strain of bacteria. That is why the DH5 strain is also included in the AdenoVator™ kit is that it allows amplification without rearrangements (does not support recombination).

In the following reference, researchers have first introduced the viral plasmid into BJ5183 cells and then electroporated the recombined transfer vector into the cells:

Zeng, M. et al. (2001). AdEasy system made easier by selecting the viral backbone plasmid proceeding homologous recombination. BioTechniques 31:260-262.

Q. What is the best way to screen for recombinant DNA made with the AdenoVator™ kit?

A. The restriction enzyme Bst XI gives a very good diagnostic pattern to screen recombinants with the AdenoVator™ system. The restriction fragments of the pAdenoVator E1/E3 plasmid are as follows:

1- 11921 bp
2- 8237 bp*
3- 5251 bp
4- 4254 bp
5- 2379 bp
6- 1399 bp

Fragment number 2 is the only one that will vary with the size of insert and from one transfer vector to another. With pAdenoVator-CMV5, it shifts up to about 11-12 kb (and higher with the insert) whereas with pShuttle, it shifts to approximately 10.5 kb. This band may sometimes be confused with band 1 but the disappearance of band 2 is a good indication of a positive recombinant. With pAdenoVator-CMV5-IRES-GFP/BFP, band 2 is cut in the IRES fragment to give a smaller shift of that band and an additional band at approximately 2 kb. This, in conjunction with the Pac I restriction pattern, makes identification of positive recombinants very straightforward.

Q. Upon screening recombinants with Pac I, some clones give a 30-35 kb band and a 3.0 kb band, others the same 30-35 kb and a 4.5 kb band. Which one is the good pattern?

A. Some recombinants might come from a recombination event between the right side homologous regions and the origins of replication instead of with the left homologous regions (see applications manual). These 2 recombinants are equally good and can very well be used but will give different restriction patterns. In our own experience, the pattern giving a band at 4.5 kb is generally the most abundant.

Q. If I have a virus expressing the GFP or the BFP, how soon after infection can I start seeing some fluorescence?

A. Infected cells should start expressing a detectable level of GFP or BFP 4-20 hours after infection. The level of expression will vary with the promoter and the cell type used; therefore, in some instances it may take up to 24 hours. The GFP, being a stable protein builds up in the cell whereas an unstable protein will be degraded in the cell. Accordingly, the concentration of the GFP will increase over time.

Q. What is an IRES sequence?

A. An IRES, or Internal Ribosomal Entry Segment, is a sequence that supports translation initiation from the second cistron in a dicistronic message. In our transfer vector pAdenoVator-CMV5-IRES-GFP or BFP, the first cistron is your gene of interest and the second cistron is the GFP. IRES elements were first identified from the encephalomyocarditis virus messages (Ghattas et al., (1991) Mol. Cell. Biol. 11:5848-5859) but were later found in some eukaryotic messages. The secondary structure of the IRES appears to be very important for its function, more important than the sequence itself.

Note that expression levels of the two genes in this dicistronic message is not equimolar; from our own experience we estimate the proportion of genes expressed to be 3:1 (1 being the gene under IRES i.e. the GFP or BFP in our construct).

See reference: Mosser, D. D., A. W. Caron, et al. (1997). "Use of a dicistronic expression cassette encoding the green fluorescent protein for the screening and selection of cells expressing inducible gene products." BioTechniques 22(1): 150-61.