Gene expression vectors are an important tool in life science research.
Most conventional vectors are used to constitutively express a transgene of interest, meaning that
the researcher will have little to no control over the duration or level of expression of
the gene.
However, such uncontrolled expression may cause cell death when studying toxic genes
or genes which are not well tolerated by the cell.
Thus, inducible expression can be useful to determine a clear genetic basis for a phenotype,
by observing the phenotype appearing and disappearing as transcription is turned "on" or "off".
For these cases, it is useful to use a gene expression vector with a promoter that only
becomes active once a given compound is added.
A number of inducible systems have been developed for use in mammalian cells, including regulation
by steroid hormones, heavy metals, heat shock, and tetracycline.
One limitation of these systems is the "leakiness", or background expression, that sometimes occurs
in the "off" state.
Another problem is that the inducers may cause unwanted effects of their own, which can cause
cytotoxicity or confound data about transgene expression.
To combat these problems, scientists at the National Research Council of Canada have developed
the Cumate Inducible System.
As the name suggests, the inducing element for this system is cumate, a small non-toxic
molecule that can be added directly to the culture medium.
Cumate is an excellent inducer since it has no significant effect on a cell's protein
expression, is inexpensive, and is not found in commonly-used cell culture reagents.
How does the cumate inducible system work?
There are three elements involved: the cumate operator sequence (also known as CuO), the
CymR repressor, and the inducer, cumate.
The cumate operator sequence is found downstream of a promoter and upstream of the transgene.
When cumate is not present, CymR binds with high affinity to the cumate operator sequence
and blocks transcription of the transgene, leaving the system in the "off" state.
However, as soon as cumate is added it associates with CymR and prevents it from binding to
the cumate operator. This switches the system "on" and allows transcription to proceed
unhindered.
Level of gene expression can be fine-tuned by adding greater or lesser amounts of cumate.
For example, it's easy to see here that the level of GFP reporter expression increases
when more cumate is added to the media.
It's also easy to turn the system "off" again, simply by performing a quick and easy
media change to remove the cumate.
The system may be turned "on" and "off" at will, as many times as desired.
abm's cumate-inducible lentiviral vectors use a CMV promoter (cumate-inducible products no longer offered), as it leads to strong
gene expression and is active in a broad range of mammalian cells.
Our system is optimized for a dual vector format, where the CymR repressor is provided
in one vector and the cumate-inducible gene of interest is in another.
The dual vector format allows large inserts to be packaged, and ensures tight regulation
of gene expression.
Our cumate-inducible cloning vectors are available with or without constitutive or inducible
reporter gene expression (cumate-inducible products no longer offered).
Constitutive GFP or RFP expression that is not controlled by cumate may be useful for
monitoring transfection efficiency and facilitating stable cell line generation.
Inducible reporter expression is useful for monitoring the efficiency of induction and
relative gene expression.
We also offer pre-made cumate-inducible lentiviral vectors and viruses for expression of any
human, mouse, or rat gene, or any or miRNA from our extensive clone collection (cumate-inducible products no longer offered).
The CymR repressor can be expressed using one of our CymR repressor vectors, or for
more convenience, you can use our pre-made CymR-expressing stable cell lines.
In conclusion, inducible gene expression is useful for:
Studying a gene which would be deleterious when constitutively expressed.
Determining a clear cause-and-effect relationship between genotype and phenotype.
The cumate-inducible system offers the following advantages over other inducible systems:
1. Tight regulation of gene expression with extremely low background expression levels.
Very little "leakage" compared to similar systems.
2. It's easy to adjust the level of gene expression by adjusting cumate concentration.
The relationship between concentration and expression is linear and titratable.
3. Gene expression is reversible and may be turned "on" and "off" as many times as required.
and 4. No special media or conditions are required during cell culture.
At abm, we offer everything you need to perform your own cumate-inducible expression experiment (cumate-inducible products no longer offered).
If you're interested, click the link in the description.
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