pFB-ERV載體描述 DNA vector-based systems that allow precise control of gene expression in vivo have become invaluable for the study of gene function in a variety of organisms, particularly when applied to the study of developmental and other biological processes for which the timing or dosage of gene expression is critical to gene function. Such systems have also been successfully used to overexpress toxic or disease-causing genes, to induce gene targeting, and to express antisense RNA. Inducible systems are currently being used by pharmaceutical companies to facilitate screening for inhibitors of clinically relevant biological pathways, and potential applications for gene therapy are being explored.
The Agilent Complete Control ecdysone-inducible plasmid vectors are based on the insect molting hormone ecdysone, which can stimulate transcriptional activation in mammalian cells harboring the ecdysone receptor protein from Drosophila melanogaster.2 The system has a number
of advantages over alternative systems. Firstly, the lipophilic nature and short in vivo half-life of the ecdysone analog ponasterone A (ponA) allows efficient penetrance into all tissues including brain, resulting in rapid and potent inductions and rapid clearance. Secondly, ecdysteroids are not known, nor are they expected, to affect mammalian physiology in any measurable way. Thirdly, the heterodimeric ponA responsive receptor and receptor DNA recognition element have been genetically altered such that trans-activation of endogenous genes by the ecdysone receptor, or of the ponA-responsive expression cassette by endogenous transcription factors, is extremely unlikely. In addition, it has been found that in the absence of inducer the heterodimer remains bound at the promoter in a complex with corepressors and histone deacetylase, and is thus tightly repressed until ligand binding, at which time high-level transcriptional activation occurs (i.e., the heterodimer is converted from a tight repressor to a transactivator). In transient assays and stable cell lines harboring receptor expression plasmids in combination with a plasmid bearing an inducible luciferase expression cassette, induction ratios of 1,000-fold have been achieved.3
A limitation to the use of plasmid-based vectors for controlled gene expression is the fact that many cell types of academic, industrial or clinical interest are difficult or virtually impossible to transfect using current transfection methods. In particular, primary human cell lines, lymphocytes, neurons and other nondividing cells are best transduced using viral delivery systems. The most popular and user-friendly of these are the retroviral vectors. Infection with retroviruses often yields transduction efficiencies close to 100%, and the proviral copy number can be easily controlled by varying the multiplicity of infection (MOI). This latter feature is particularly important for inducible systems, for which low basal expression and high induction ratios are affected by copy number. Thus infection of the target cell with virus at an optimal MOI should yield a high frequency of clones capable of mediating desirable expression profiles without exhaustive colony screening.
With the vectors pFB-ERV and pCFB-EGSH, we have adapted the ecdysone inducible components of the Complete Control System for retroviral delivery. Used together, we have attained induction ratios of >1,000-fold with these vectors in tissue culture cells.
OVERVIEW OF ECDYSONE-REGULATABLE GENE EXPRESSION
The ecdysone receptor (EcR) is a member of the retinoid-X-receptor (RXR) family of nuclear receptors and is composed of three domains: an N-terminal activation domain (AD), a central DNA-binding domain (DBD), and a C-terminal ligand-binding and dimerization domain (LBD). In insect cells, EcR and the nuclear receptor ultraspiracle (USP) form a promoterbound heterodimer, which regulates transcription (see Figure 1). In the absence of ecdysone, the receptor heterodimer binds to corepressors and tightly represses transcription.4
When ecdysone binds to the EcR LBD, the corepressors are released, coactivators are recruited to the complex, and transcriptional activation is enabled.
In mammalian cells harboring the EcR gene, EcR heterodimerizes with RXR, the mammalian homologue of USP. The EcR–RXR heterodimer binds to multiple copies of the ecdysone-responsive element (EcRE), and in the absence of ponA, represses transcription of an expression cassette. When ponA binds to the receptor, the receptor complex activates transcription of a reporter gene or a gene of interest. To avoid pleiotropic interactions with endogenous pathways in mammalian host cells, both the EcRE recognition sequence and the EcR protein were modified.
The EcRE sequence was modified to create a synthetic recognition site that does not bind any endogenous transcription factors. The wild-type EcRE sequence consists of two inverted repeat sequences separated by a single nucleotide: AGTGCA N TGCACT. The EcRE sequence was changed to AGTGCA N1 TGTTCT (and renamed E/GRE). Recognition of the synthetic E/GRE recognition sequence by either a steroid receptor or a wild-type RXR heterodimer receptor is extremely unlikely, as these receptors recognized only the wild-type perfect inverted repeat. The E/GRE recognition sequence has imperfect inverted half sites separated by one nucleotide. A wild-type RXR heterodimer requires single nucleotide separation of the inverted repeats, and the majority bind to direct repeats rather than inverted repeats (EcRE is an exception).
The EcR protein was modified to create a synthetic ecdysone-binding receptor that does not transactivate any host genes. Three amino acids in the EcR DBD were mutated to change its DNA-binding specificity to that of the glucocorticoid receptor (GR), which recognizes the half-site AGAACA.2 Like all steroid receptors and unlike RXR receptors, the GR protein homodimerizes and recognizes two inverted repeat sequences separated by three nucleotides. The GR–EcR fusion protein (GEcR) retains the ability to dimerize with RXR and activate, with ponA-dependence, reporter genes that contain the synthetic E/GRE recognition sequence.
The GEcR receptor was further modified by replacing the EcR AD with the more potent VP16 AD. The result of all the modifications is the synthetic ecdysone-binding receptor VgEcR. VgEcR is a fusion of the ligand-binding and dimerization domain of the D. melanogaster ecdysone receptor, the DNA-binding domain of the glucocorticoid receptor, and the transcription activation domain of herpes simplex virus (HSV) VP16
OVERVIEW OF REPLICATION-DEFECTIVE RETROVIRAL GENE TRANSFER SYSTEMS
Non-replicating retroviral vectors contain all of the cis elements required for transcription of mRNA molecules encoding a gene of interest, and packaging of these transcripts into infectious virus particles (Figure 2). The vectors are typically comprised of an E. coli plasmid backbone containing a pair of 600 base pair viral long terminal repeats (LTRs) between which the gene of interest is inserted. The LTR is divided into 3 regions. The U3 region contains the retroviral promoter/enhancer. The U3 region is flanked in the 3′ direction by the R region, which contains the viral polyadenylation signal (pA), followed by the U5 region which, along with R, contains sequences that are critical for reverse transcription. Expression of the viral RNA is initiated within the U3 region of the 5′ LTR and is terminated in the R region of the 3′ LTR. Between the 5′ LTR and the coding sequence for the gene of interest resides an extended version of the viral packaging signal (ψ+), which is required in cis for the viral RNA to be packaged into virion particles.
In order to generate infectious virus particles that carry the gene of interest, specialized packaging cell lines have been generated that contain chromosomally integrated expression cassettes for viral Gag, Pol and Env proteins, all of which are required in trans to make virus. The gag gene encodes internal structural proteins, pol encodes reverse transcriptase (RT) and integrase, and the env gene encodes the viral envelope protein, which resides on the viral surface and facilitates infection of the target cell by direct interaction with cell type-specific receptors; thus the host range of the virus is dictated not by the DNA vector but by the choice of the env gene used to construct the packaging cell. The packaging cell line is transfected with the vector DNA, and at this point either stable viral producer cell lines may be selected (providing the vector has an appropriate selectable marker), or mRNAs that are transiently transcribed from the vector are encapsidated and bud off into the cell supernatant. These supernatants are collected, and used to infect target cells. Upon infection of the target cell, the viral RNA molecule is reverse transcribed by RT (which is present in the virion particle), and the cDNA of the gene of interest, flanked by the LTRs, is integrated into the host DNA. Because the vector itself carries none of the viral proteins, once a target cell is infected the LTR expression cassette is incapable of proceeding through another round of virus production. Recent advances in transfection technology have allowed the production of high titer viral supernatants following transient cotransfection of the viral vector together with expression vectors encoding the gag, pol and env genes (Figure 2),5, 6 obviating the need for the production and maintenance of stable packaging cell lines. For example, Agilent pVPack gag-pol and env-expressing packaging vectors consistently give rise to titers of >107 infectious units (IU)/ml when cotransfected with the pFB-hrGFP control vector (Agilent Catalog #240027), using a 293-derived cell line for virus production.
Description of the Vectors
The pFB-ERV vector was derived from the high-titer MoMLV vector pFBNeo5 for efficient delivery of the ecdysone receptor proteins VgEcR and RXR (Figure 3). In the vector pFB-ERV the ecdysone receptor and the neomycin-resistance open reading frame (ORF) are expressed from a tricistronic message with the neomycin resistance ORF expressed at the end of the message. Thus, maintenance of infected cell lines in G418 ensures expression of the transcript encoding the receptor genes. The tricistronic transcript is expressed from the CMV promoter, which is flanked by unique EcoR I and Fse I sites so that a cell type-specific promoter of interest may be substituted. The viral promoter within the 3′ LTR has been deleted to make this a self-inactivating (SIN) vector. Upon infection and chromosomal integration into the target cell genome, the SIN deletion is transferred to the 5′ LTR, resulting in an integrated expression cassette in which only the CMV promoter is active. Cells containing an estimated single integrated viral expression cassette can be selected in as high as 1 mg/ml G418, although 600 μg/ml is routinely used.
The vector pCFB-EGSH contains an ecdysone-inducible expression cassette inserted between the viral LTRs in the antisense orientation relative to that for the viral promoter (see Figure 4). The U3 promoter within the 5′ LTR of the vector has been replaced with the CMV promoter to increase production of viral RNA in packaging cells, thereby increasing the titer of the viral supernatants. Potential interference from the proviral 5′ LTR is obviated due to the SIN deletion. The inducible expression cassette contains a multiple cloning site that contains three contiguous copies of the HA epitope(3× HA) positioned for fusion at the C-terminus of the protein of interest. A second expression cassette in which the hygromycin-resistance gene is expressed from the TK promoter is located downstream (relative to transcription from the LTRs) of the inducible cassette. A pBR322 origin and ampicillin-resistance gene allow pCFB-EGSH to be propagated in prokaryotes.
The pCFB-EGSH-Luc vector contains the luciferase reporter gene and is intended for use as a positive control vector to test the expression of the VgEcR and RXR receptors in pFB-ERV-containing cell lines. The pCFB-EGSH-Luc vector is derived from the pCFB-EGSH vector and has the luciferase gene inserted in the MCS. The pCFB-EGSH-Luc vector does not contain the HA epitope sequence. pFB-ERV載體限制性酶切位點 pFB-ERV, 11067 bp version 011006
Enzymes with 1-10 cleavage sites:
#sites -- Bp position of recognition site --
AarI 3 5297, 6078, 7229
AatII 7 978, 2200, 2253, 2336, 2522
3692, 10993
Acc65I 8 647, 1847, 2966, 3609, 4792
5409, 7341, 8593
AccI 6 1968, 2058, 3212, 3762, 6234
8953
AccIII 1 8514
AclI 3 4990, 10300, 10673
AflII 3 202, 1265, 8466
AflIII 9 164, 2820, 3249, 5283, 5458
7215, 7390, 8545, 9182
AgeI 2 1841, 6125
AhdI 6 687, 733, 1274, 8633, 8679
10070
AleI 2 5607, 6402
Alw44I 6 1054, 5445, 7377, 8998, 9496
10742
AlwNI 3 323, 398, 9593
ApaI 5 1238, 5081, 5754, 5787, 7013
ApoI 4 87, 1128, 2064, 2986
AscI 2 3231, 8537
AseI 2 2085, 10246
AvaI 10 577, 610, 643, 1241, 2019
4259, 4340, 5700, 8556, 8589
BamHI 4 3167, 6272, 6425, 6539
BbeI 7 615, 1656, 4376, 6598, 6939
7704, 8561
BbsI 10 2714, 3947, 4148, 5101, 5201
5567, 7033, 7133, 7499, 11060
BbvCI 6 453, 4448, 4454, 4526, 4538
6744
BciVI 7 656, 1987, 3038, 7913, 8602
9391, 10918
BclI 1 8508
BfrBI 1 8519
BglI 10 2163, 2285, 2356, 3926, 4122
4859, 5264, 5906, 7196, 10189
BglII 2 1678, 4266
BlnI 3 5119, 7051, 7542
BlpI 4 4349, 5850, 6283, 6817
BmgBI 4 2774, 5508, 6562, 7440
BmrI 6 2373, 5318, 7250, 7644, 8928
10120
BmtI 6 6, 16, 26, 197, 4772
8461
BpmI 7 1799, 4049, 4592, 6277, 6934
8414, 10160
Bpu10I 10 337, 412, 453, 1548, 4448
4454, 4526, 4538, 5730, 6744
BpuEI 8 5370, 5920, 7302, 8096, 9288
9550, 9827, 10695
BsaAI 8 2001, 2417, 5282, 6316, 7214
8007, 8524, 8934
BsaBI 1 2807
BsaI 10 694, 715, 782, 1414, 1802
6310, 8640, 8661, 8728, 10142
BsgI 3 5668, 6184, 6559
BsiWI 1 4187
BsmBI 8 976, 1093, 1337, 1396, 1582
4337, 4630, 8831
BsmI 7 3317, 4894, 5114, 5147, 5712
7046, 7079
BspHI 3 9902, 10910, 11015
BspMI 7 3085, 3951, 5298, 6079, 7230
7601, 7964
BsrDI 6 3420, 5072, 7004, 7931, 10129
10311
BsrGI 3 1541, 3252, 6017
BssHII 6 563, 3232, 3551, 6681, 8102
8538
BssSI 4 8297, 9355, 10739, 11046
Bst1107I 1 8953
BstAPI 4 322, 397, 4392, 9000
BstEII 2 1346, 4961
Bsu36I 3 1276, 5939, 5996
BtsI 4 4015, 4124, 10468, 10496
ClaI 2 5534, 7466
DraI 3 9939, 9958, 10650
DraIII 3 1874, 5326, 7258
DrdI 4 6454, 7726, 8871, 9284
EagI 7 961, 2679, 3311, 4040, 6728
6962, 7611
EcoICRI 3 580, 3403, 5733
EcoNI 2 1647, 4048
EcoRI 1 2064
EcoRV 2 308, 383
FseI 1 2676
FspI 3 6059, 7805, 10295
HincII 5 2058, 3663, 4551, 6234, 10614
HindIII 3 2957, 5192, 7124
KasI 7 615, 1656, 4376, 6598, 6939
7704, 8561
KpnI 8 647, 1847, 2966, 3609, 4792
5409, 7341, 8593
MluI 1 8545
MmeI 10 684, 1383, 3823, 4811, 4936
5336, 7268, 8630, 9372, 9556
MscI 7 827, 1368, 1668, 3537, 6158
6548, 7785
MunI 2 11, 21
NaeI 5 2677, 4388, 4735, 6837, 8205
NarI 7 615, 1656, 4376, 6598, 6939
7704, 8561
NcoI 7 2439, 2688, 5651, 5783, 6161
7560, 8137
NdeI 4 1664, 1672, 2312, 9004
NgoMIV 5 2677, 4388, 4735, 6837, 8205
NheI 6 6, 16, 26, 197, 4772
8461
NotI 1 6961
NruI 2 3004, 3885
NsiI 1 8519
PciI 6 164, 2820, 3249, 5458, 7390
9182
PfoI 6 771, 2848, 3327, 6275, 8717
8826
PmlI 3 2001, 5282, 7214
PpuMI 8 499, 1477, 1925, 3082, 4723
5924, 6065, 8530
PshAI 2 1015, 2731
PspOMI 5 1238, 5081, 5754, 5787, 7013
PstI 4 1178, 1360, 7754, 10316
PvuI 4 1034, 5532, 7464, 10442
PvuII 6 286, 361, 4541, 5707, 6021
7809
RsrII 2 4035, 8220
SacI 3 580, 3403, 5733
SacII 3 151, 4431, 4677
SalI 2 2058, 6234
SanDI 1 8530
SapI 5 3070, 3202, 8054, 8264, 9059
ScaI 2 6134, 10553
SexAI 3 1474, 4663, 6458
SfcI 10 182, 1178, 1360, 5216, 7148
7754, 8446, 9447, 9638, 10316
SfoI 7 615, 1656, 4376, 6598, 6939
7704, 8561
SmaI 5 643, 1241, 2019, 5700, 8589
SnaBI 2 2417, 8524
SpeI 1 897
SphI 3 2788, 3502, 8106
SrfI 1 1240
SspI 3 3532, 3877, 10877
StuI 2 3813, 6773
TatI 10 1541, 2296, 2376, 2409, 2460
3252, 6017, 6134, 8988, 10553
TfiI 8 2837, 3469, 3911, 4493, 4603
8190, 8324, 9157
Tsp45I 9 1282, 1491, 6364, 7826, 8132
8837, 8932, 10332, 10543
Tth111I 8 633, 1473, 3992, 6358, 6457
7820, 8579, 8926
Van91I 2 5415, 7347
XbaI 3 464, 2037, 2972
XcmI 2 5741, 5813
XhoI 2 4259, 4340
XmaI 5 643, 1241, 2019, 5700, 8589
XmnI 3 5176, 7108, 10670
ZraI 7 978, 2200, 2253, 2336, 2522
3692, 10993
Enzymes that do NOT cut molecule:
AsiSI BstBI BstXI Eco47III FspAI
HpaI PacI PmeI PsiI SbfI
SfiI SgrAI SwaI
