Adeno-Quest™

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. 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 the subtype of the Ad5 viral backbone used for the Adeno-Quest™ constructs?

A. The viral backbone we use for the Adeno-Quest™ constructs is an Ad5 subtype delta L327, with a 1.89 kb XbaI fragment deleted that covers almost the entire E3 region from 28,593-30,471 (78.5-84.3 Map units).
The backbone is further E1 deleted from nucleotides 358-3,332 (numbers are based on Ad5 wt sequence, map units 1.4 to 9.4). This removes nearly all the E1 sequences between the viral packaging domain and the coding sequence for pIX.

Q. How strong is the Ad MLP (Major Late Promoter), or BM5 promoter, compared to the CMV5 promoter?

A. Although the MLP is active in the early phase of the infection, its transcriptional activity is increased 30-50 fold during the late phase i.e. in 293 cells only. In non-permissive cells, its strength compares with a minimal CMV promoter.

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 pQBI-AdCMV5-IRES-GFP, the first cistron is gene X (i.e. your favorite gene) 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).

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.