pRL-null

The pRL-null Vector(a,b) (Figure 1) is intended for use in constructing a control
reporter vector that may be used in combination with any experimental reporter vector
to cotransfect mammalian cells. All of Promega’s pRL Reporter Vectors contain a
cDNA(b) (Rluc) encoding Renilla luciferase, which was originally cloned from the
marine organism Renilla reniformis (sea pansy; 1). As described below, the Renilla
luciferase cDNA contained within the pRL Vectors has been modified slightly to
provide greater utility.
The pRL-null Vector contains no enhancer or promoter elements. Rather, it contains
a multiple cloning region upstream of Rluc to allow for the cloning of any desired regulatory
element(s) to drive expression of Renilla luciferase. Renilla luciferase is a
36kDa monomeric protein that does not require post-translational modification for
activity (2). Therefore, like firefly luciferase, the enzyme may function as a genetic
reporter immediately following translation. For information about the use of this plasmid
in conjunction with a reporter vector containing the firefly luciferase gene, refer
to the Dual-Luciferase Reporter Assay System(c,d) Technical Manual (#TM040).
The pRL Vectors are isolated from a dam–/dcm– E. coli K host strain, allowing
digestion with restriction enzymes that are sensitive to dam and dcm methylation.
The GenBank/EMBL Accession Number for the pRL-null Vector is AF025844.

Features of the pRL-null Vector

A. Multiple Cloning Region
The pRL-null Vector contains a multiple cloning region positioned immediately
upstream of the chimeric intron and Renilla luciferase reporter gene (Figure 2).
To aid in devising cloning strategies, Table 1 summarizes the types of DNA ends
generated from restriction endonuclease digestion within the multiple cloning
region as well as the compatibility of those ends with the ends of DNA fragments
generated by heterologous restriction enzymes.

B. Chimeric Intron
Downstream of the multiple cloning region of the pRL-null Vector is a chimeric
intron comprised of the 5′-donor splice site from the first intron of the human
β-globin gene, and the branch and 3′-acceptor splice site from an intron preceding
an immunoglobulin gene heavy chain variable region (3). The sequences of
the donor and acceptor splice sites, along with the branchpoint site, have been
modified to match the consensus sequences for optimal splicing (4).
Transfection studies have demonstrated that the presence of an intron flanking a
cDNA insert frequently increases the level of gene expression (5–8). In the
pRL-null Vector the intron is positioned 5′ to Rluc to minimize the utilization of
cryptic 5′-donor splice sites that may reside within the reporter gene sequence (9).

C. T7 Promoter
A T7 promoter is located downstream of the chimeric intron and immediately
precedes the Rluc reporter gene. This T7 promoter can be used to synthesize
RNA transcripts in vitro using T7 RNA Polymerase (Cat.# P2075). T7 RNA
Polymerase can also be used to synthesize active Renilla luciferase in a cell-free
coupled eukaryotic in vitro transcription/translation reaction (e.g., Promega’s
TNT Reticulocyte Lysate(c,e,f,g) [Cat.# L4610], TNT T7 Coupled Wheat Germ
Extract(c,e,f,g) [Cat.# L4140] or TNT T7 Quick Coupled Transcription/Translation
(c,e,f,g,h) [Cat.# L1170] Systems).

D. Renilla Luciferase Reporter Gene (Rluc)
The Renilla luciferase cDNA inserted into all of the pRL Vectors is derived from
the anthozoan coelentrate Renilla reniformis (1) but contains nucleotide changes
that were engineered during the construction of the individual vectors. The following
bases were altered in the pRL-null Vector: base 539 (T→C), to eliminate
an internal Bgl II site; base 1082 (T→C), to eliminate an internal BamH I site;
base 1115 (C→T), to eliminate internal Nar I, Kas I, Ban I and Acy I sites. These
nucleotide substitutions do not alter the amino acid sequence of the encoded
Renilla luciferase reporter enzyme.

E. SV40 Late Polyadenylation Signal
Polyadenylation signals cause the termination of transcription by RNA polymerase
II and signal the addition of approximately 200–250 adenosine residues
to the 3′-end of the RNA transcript (10). Polyadenylation has been shown to
enhance RNA stability and translation (11,12). The late SV40 polyadenylation
signal, which is extremely efficient and has been shown to increase the steadystate
level of RNA approximately 5-fold over the early SV40 polyadenylation signal
(13), has been positioned 3′ to the Rluc gene in the pRL-null Vector to
increase the level of Renilla luciferase expression.

IV. Transfection of Mammalian Cells with the pRL-null Vector

The pRL-null Vector, once it has been modified to contain appropriate genetic regulatory
domains, may be used in combination with any experimental reporter vector to
cotransfect mammalian cells. However, it is important to realize that trans effects
between promoters on cotransfected plasmids can potentially affect reporter gene
expression (14). Primarily this is of concern when either the control or experimental
reporter vector, or both, contain very strong promoter/enhancer elements. The
occurrence and magnitude of such effects will depend on several factors: i) the combination
and activities of the genetic regulatory elements present on the cotransfected
vectors; ii) the relative ratio of experimental vector to control vector introduced
into the cells; and iii) the cell type transfected.
To help ensure independent genetic expression between experimental and control
reporter genes, preliminary cotransfection experiments should be performed to optimize
both the amount of vector DNA and the ratio of the coreporter vectors added to
the transfection mixture. Similar to the firefly luciferase assay, the Renilla luciferase
assay is extremely sensitive, providing accurate measurement of ≤10 femtograms of
Renilla luciferase, with linearity over seven orders of enzyme concentration.
Therefore, it is possible to use relatively small quantities of the pRL-null Vector to
provide low-level, constitutive coexpression of Renilla luciferase control activity.
The pRL-null Vector, once genetic regulatory domains have been added, can be
used for both transient and stable expression of Renilla luciferase. For stable expression,
the pRL-null Vector must be cotransfected with an expression vector containing
a selectable gene in mammalian cells. Transfection of DNA into mammalian cells
may be mediated by cationic lipids (15,16), calcium phosphate (17,18), DEAEDextran
(19–21), polybrene-DMSO (22,23), or electroporation (24,25).
Transfection systems based on cationic lipid compounds (TransFast Reagent(i),
Tfx Reagents(j) and Transfectam Reagent(k)), calcium phosphate and DEAEDextran
are available from Promega. For more information and a protocol for the
Transfectam Reagent, please request the Transfectam Reagent Technical Bulletin
(#TB116) and for the TransFast Reagent, please request the TransFast
Transfection Reagent Technical Bulletin (#TB260). Protocols for the use of the
Tfx Reagents can be found in the Tfx-10, Tfx-20 and Tfx-50 Reagents for
the Transfection of Eukaryotic Cells Technical Bulletin (#TB216). For transfection procedures
using calcium phosphate or DEAE-Dextran, please request the ProFection
Mammalian Transfection Systems Technical Manual (#TM012).