Q. Will extra bases in the 5' and 3' UTRs (Untranslated
regions) of my gene cloned into a transfer vector affect protein
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
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.
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.
Q. Is it possible to first transform the BJ5183 with pAdenoVator
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
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