pRL-TK Vector质粒图谱及其说明书

pRL-TK Vector base 1264 (T→C) to eliminate an internal BglII site,

base 1807 (T→C) to eliminate internal BamHI site,

base 1840 (C→T) to eliminate internal NarI, KasI, BanI

and AcyI sites.

1. Description

The pRL Reporter Vectors contain a cDNA (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 Reporter Vectors has been modified slightly to provide greater utility.

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 Technical Manual (#TM040) or the Dual-Glo® Luciferase Assay System Technical Manual (#TM058).

To avoid DNA methylation, all pRL Reporter Vectors are isolated from a

dam–/dcm– E. coli K host strain. If you use methylation-sensitive restriction

enzymes (e.g., BclI, ClaI, MboI, TaqI or XbaI), continue to propagate the pRL

Reporter Vectors in a host strain with the same genetic background.

2. Features of the pRL Reporter Vectors

The pRL-TK Vector(a) is intended for use as an internal control reporter vector

and may be used in combination with any experimental reporter vector to

co-transfect mammalian cells. The pRL-TK Vector contains the herpes

simplex virus thymidine kinase (HSV-TK) promoter to provide low to

moderate levels of Renilla luciferase expression in co-transfected

mammalian cells.

A Chimeric Intron

Downstream of either the constitutive promoter regions (CMV, TK or SV40) or

the multiple cloning site of the pRL Vectors 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 Reporter Vectors, 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).

B 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., our TNT®

T7 Coupled Reticulocyte Lysate [Cat.# L4610], TNT® T7 Coupled Wheat Germ

Extract [Cat.# L4140] or TNT® T7 Quick Coupled Transcription/Translation

[Cat.# L1170] Systems).

Note: The T7 Promoter Primer offered by Promega (Cat.# Q5021) cannot be

used for sequencing this vector because of a mismatch between the 3´ end of

the primer and the vector DNA.

C 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 is extremely efficient and has been shown to increase

the steady-state level of RNA approximately fivefold more than the early SV40

polyadenylation signal (13). This poly(A) signal has been positioned 3´ to the

Rluc gene in the pRL Reporter Vectors to increase the level of Renilla luciferase

expression.