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Which pRL Vector should I use for a Dual-Luciferase® Reporter Assay(a,b)?
Promega offers four high copy number pRL Vectors(c,d)
driving expression of Renilla luciferase(c) (each
in a pUC-based, high copy number expression plasmid). pRL-TK Vector
(Cat.# E2241)
contains the thymidine kinase promoter; pRL-SV40 Vector (Cat.# E2231), the SV40
promoter; and pRL-CMV Vector(e) (Cat.#
E2261), the cytomegalovirus promoter, and pRL-null Vector
(Cat.# E2271), which
lacks apparent enhancer/promoter elements. The TK, SV40 and CMV regulatory elements are
all derived from pathogenic mammalian viruses and have been reported as being expressed in
most mammalian cell types; however, exact expression levels achieved are cell-specific.
In order to choose the suitable co-reporter vector for normalization of transfection
variability, a few factors must be considered:
- Relative light units (RLU) from expression of the co-reporter. The
co-reporter signal has to be high enough to obtain reliable readings and better
signal-to-noise ratios.
- Promoter cross talk or interference. Usually co-reporter expression is
driven by a viral promoter to achieve constitutive and high expression. A strong promoter
may cause a so-called “squelching” effect--soaking up general transcription
factors, or it may specifically interfere with the test promoter by competing for the same
factor(s) that is(are) required for transcription for both promoters.
- Undesirable regulation of co-reporter expression by experimental stimulus.
Analysis of a test promoter can involve looking into its regulation under different
experimental stimuli. There is a possibility that the co-reporter expression is also
influenced by the same treatment making it unreliable as the internal control. In such
instances, viral promoter activity of the co-reporter could be affected by the
experimental stimulus; or spurious expressions occur as a result of cryptic
transcriptional regulatory sites in the plasmid, which become functional under certain
experimental conditions.
To provide customers with a choice of co-reporter vectors that best fit their
needs and to address specific concerns they might have, Promega offers an array of
luciferase vectors, each with different characteristics. pRL-TK usually yields
low-to-moderate levels of expression in many cell lines. pRL-TK promoter activity is not
so robust as CMV or SV40 and it is also less likely to be affected by other experimental
treatments. As a result, pRL-TK is an ideal initial vector to choose if no other vectors
have been identified as suitable. For those transfecting cell lines in which pRL-TK
expression is rather poor or transfection efficiency is low, pRL-SV40 Vector is
recommended. It has shown good expression in many cell lines while still possessing fewer
binding sites for transcriptional regulators as compared to CMV. Therefore, this vector is
particularly useful if the signal level generated by the co-reporter is a concern. We
suggest resarchers use the pRL-CMV Vector when a TK or SV40 promoter does not generate
adequate signals. But be advised that the robust and promiscuous activity of the CMV
promoter could cause promoter interference or undesirable regulation of expression under
experimental stimuli. The CMV enhancer/promoter is notorious for "cross-talk"
with other promoters as it contains many transcription factor binding sites (e.g., two
AP2, four NF-kappaB, and five CREB binding sites). Due to the presence of these
factor-binding sites, CMV promoter activity is also likely to be affected by some
experimental stimuli. For those studying a test promoter under different signal
transduction pathways and are particularly concerned about, or have found that, other
viral promoter activities are influenced by the experimental conditions they are using,
pRL-null Vector could be a good choice. Even without an apparent promoter, this vector
does express luciferase in some cell lines (1) and has been shown to be immune to
regulation by signal cascades induced by activated Ras or activated MEKK1. It is a
valuable adjunct to studies investigating transcriptional activation mediated by Ras or
other non-nuclear oncogenes. The disadvantage of pRL-null Vector is that its expression
level may not be desirable in some cell lines.
Although the above provides some guidance as to which co-reporter vector to choose, due to
the complexity of transcriptional events in a specific cellular context, testing
different co-reporter vectors is recommended before the best one is found for your
research purpose.
Reference
- Behre, G. et al. (1999) Use of a promoterless Renilla luciferase
vector
as an internal control plasmid for transient co-transfection assays of
Ras-mediated transcription activation. BioTechniques 26, 24.
(a)U.S. Pat. No. 5,744,320.
(b)U.S. Pat. No. 5,283,179, Australian Pat. No. 649289
and other patents. Certain applications of this product may require licenses from others.
(c)Licensed under U.S. Pat. Nos. 5,292,658, 5,418,155
and other patents.
(d)Certain applications of this product may require
licenses from others.
(e)The CMV promoter and its use are covered under U.S.
Pat. Nos. 5,168,062 and 5,385,839 owned by the University of Iowa Research Foundation,
Iowa City, Iowa, and licensed FOR RESEARCH USE ONLY. Commercial users must obtain a
license to these patents directly from the University of Iowa Research Foundation.
Dual-Luciferase is a trademark of Promega Corporation and is registered
with the U.S. Patent and Trademark Office. |
