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基本信息

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載體描述

pRL系列是野生型海腎螢光素酶(Rluc)報(bào)告基因?qū)φ蛰d體。pRL載體提供組成型表達(dá)的海腎螢光素酶，可與一個(gè)螢火蟲螢光素酶載體聯(lián)合共轉(zhuǎn)染哺乳動(dòng)物細(xì)胞。海腎螢光素酶的表達(dá)為實(shí)驗(yàn)的螢火蟲螢光素酶報(bào)告基因正態(tài)化提供內(nèi)對(duì)照值。

The pRL-TK Vector 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. This is primarily 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 type of cell transfected.Features of the pRL-TK Vector

A. TK Promoter Region

The pRL-TK Vector contains the herpes simplex virus thymidine kinase promoter region upstream of Rluc. The HSV-TK promoter provides low-level, constitutive expression in cells of both embryonal and mature mammalian tissues.

B. Chimeric Intron

Downstream of the TK promoter region of the pRL-TK Vector is a chimeric intron comprised of the 5′-donor splice site from the first intron of the humanβ-globin gene, and the branch and 3′-acceptor splice site from an intron preceding an immunoglobulin gene heavy chain variable region (5). 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 (6).

Transfection studies have demonstrated that the presence of an intron flanking a cDNA insert frequently increases the level of gene expression (7–10). In the pRL-SV40 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.

C. T7 Promoter

A T7 promoter is located downstream of the chimeric intron, immediately preceding 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.

D. Renilla Luciferase Reporter Gene (Rluc)

The Renilla luciferase cDNA inserted into all of the pRL Vectors is derived from the anthozoan coelenterate Renilla reniformis but contains nucleotide changes that were engineered during the construction of the individual vectors.

The following bases were altered in the pRL-TK Vector: base 1264 (T→C), to eliminate an internal Bgl II site; base 1807 (T→C), to eliminate internal BamH I site; base 1840 (C→T), to eliminate internal Nar I, Kas K, Ban K 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.

Polyadenylation has been shown to enhance RNA stability and translation. The late SV40 polyadenylation signal, which is extremely efficient and has been shown to increase the steadystate level of RNA approximately five-fold more than the early SV40 polyadenylation signal, has been positioned 3′to the Rluc gene in the pRL-TK Vector to increase the level of Renilla luciferase expression.

載體序列

ORIGIN

        1 agatctaaat gagtcttcgg acctcgcggg ggccgcttaa gcggtggtta gggtttgtct

       61 gacgcggggg gagggggaag gaacgaaaca ctctcattcg gaggcggctc ggggtttggt

      121 cttggtggcc acgggcacgc agaagagcgc cgcgatcctc ttaagcaccc ccccgccctc

      181 cgtggaggcg ggggtttggt cggcgggtgg taactggcgg gccgctgact cgggcgggtc

      241 gcgcgcccca gagtgtgacc ttttcggtct gctcgcagac ccccgggcgg cgccgccgcg

      301 gcggcgacgg gctcgctggg tcctaggctc catggggacc gtatacgtgg acaggctctg

      361 gagcatccgc acgactgcgg tgatattacc ggagaccttc tgcgggacga gccgggtcac

      421 gcggctgacg cggagcgtcc gttgggcgac aaacaccagg acggggcaca ggtacactat

      481 cttgtcaccc ggaggcgcga gggactgcag gagcttcagg gagtggcgca gctgcttcat

      541 ccccgtggcc cgttgctcgc gtttgctggc ggtgtccccg gaagaaatat atttgcatgt

      601 ctttagttct atgatgacac aaaccccgcc cagcgtcttg tcattggcga attcgaacac

      661 gcagatgcag tcggggcggc gcggtcccag gtccacttcg catattaagg tgacgcgtgt

      721 ggcctcgaac accgagcgac cctgcagcga cccgcttaaa agcttgattc ttctgacaca

      781 acagtctcga acttaagctg cagaagttgg tcgtgaggca ctgggcaggt aagtatcaag

      841 gttacaagac aggtttaagg agaccaatag aaactgggct tgtcgagaca gagaagactc

      901 ttgcgtttct gataggcacc tattggtctt actgacatcc actttgcctt tctctccaca

      961 ggtgtccact cccagttcaa ttacagctct taaggctaga gtacttaata cgactcacta

     1021 taggctagcc accatgactt cgaaagttta tgatccagaa caaaggaaac ggatgataac

     1081 tggtccgcag tggtgggcca gatgtaaaca aatgaatgtt cttgattcat ttattaatta

     1141 ttatgattca gaaaaacatg cagaaaatgc tgttattttt ttacatggta acgcggcctc

     1201 ttcttattta tggcgacatg ttgtgccaca tattgagcca gtagcgcggt gtattatacc

     1261 agaccttatt ggtatgggca aatcaggcaa atctggtaat ggttcttata ggttacttga

     1321 tcattacaaa tatcttactg catggtttga acttcttaat ttaccaaaga agatcatttt

     1381 tgtcggccat gattggggtg cttgtttggc atttcattat agctatgagc atcaagataa

     1441 gatcaaagca atagttcacg ctgaaagtgt agtagatgtg attgaatcat gggatgaatg

     1501 gcctgatatt gaagaagata ttgcgttgat caaatctgaa gaaggagaaa aaatggtttt

     1561 ggagaataac ttcttcgtgg aaaccatgtt gccatcaaaa atcatgagaa agttagaacc

     1621 agaagaattt gcagcatatc ttgaaccatt caaagagaaa ggtgaagttc gtcgtccaac

     1681 attatcatgg cctcgtgaaa tcccgttagt aaaaggtggt aaacctgacg ttgtacaaat

     1741 tgttaggaat tataatgctt atctacgtgc aagtgatgat ttaccaaaaa tgtttattga

     1801 atcggaccca ggattctttt ccaatgctat tgttgaaggt gccaagaagt ttcctaatac

     1861 tgaatttgtc aaagtaaaag gtcttcattt ttcgcaagaa gatgcacctg atgaaatggg

     1921 aaaatatatc aaatcgttcg ttgagcgagt tctcaaaaat gaacaataat tctagagcgg

     1981 ccgcttcgag cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg

     2041 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt

     2101 ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag

     2161 ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg taaaatcgat

     2221 aaggatccag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc

     2281 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa

     2341 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt

     2401 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct

     2461 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc

     2521 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta

     2581 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac

     2641 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc

     2701 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac

     2761 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg

     2821 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac

     2881 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc

     2941 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt

     3001 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga

     3061 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc

     3121 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag

     3181 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca

     3241 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc

     3301 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca

     3361 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc

     3421 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta

     3481 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt

     3541 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc

     3601 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg

     3661 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg

     3721 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag

     3781 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc

     3841 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat

     3901 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg

     3961 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc

     4021 tggccttttg ctcacatggc tcgac

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