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:

1. 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.
2. 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.
3. 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

1. 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.