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pFB-ERV

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  • 产品名称:pFB-ERV
  • 产品型号:
  • 产品展商:HZbscience
  • 产品文档:无相关文档
  • 发布时间:2018-07-15
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简单介绍
pFB-ERV的各批次质粒菌株发货前均经过严格的多重验证,如存在质量问题,请在收到产品的三个月内通知我司。收到pFB-ERV后请短暂离心,取2μl转化至对应感受态中,挑取单克隆重新提取质粒后使用。
产品描述

pFB-ERV载体基本信息

载体名称: pFB-ERV
质粒类型: 逆病毒载体;双顺反子载体
高拷贝/低拷贝: 低拷贝
克隆方法: 限制性内切酶,多克隆位点
启动子: CMV
载体大小: 11067 bp
5' 测序引物及序列: --
3' 测序引物及序列: --
载体标签: --
载体抗性: Kanamycin.html' target='_blank'>卡那霉素
筛选标记: 新霉素(Neomycin)
克隆菌株: DH5α 等
宿主细胞(系): 常规细胞系(293、CV-1、CHO等)
备注: --
产品目录号: 217564
稳定性: 稳表达
组成型/诱导型: 组成型
病毒/非病毒: 逆转录病毒

pFB-ERV载体质粒图谱和多克隆位点信息

pFB-ERV



pFB-ERV

pFB-ERV载体简介

pFB-ERV载体描述 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载体限制性酶切位点 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 

pFB-ERV载体序列

hz-4696R AEV polyprotein  禽脑脊髓炎病毒AEV抗体
hz-4698R EBNA 3B  EB病毒核抗原-3B抗体
hz-1164R ADFP  脂肪组织分化相关蛋白抗体
hz-4700R LMP2A  EB病毒LMP-2A蛋白抗体
hz-5851R ADAM2  去整合素样金属蛋白酶2抗体
hz-4705R MLCK  肌球蛋白轻链激酶抗体
hz-4706R Acetyl-Histone H4(K5)  乙酰化组蛋白H4抗体
hz-4707R Acetyl-Histone H4(K16)  乙酰化组蛋白H4(K16)抗体
hz-4708R AFP  甲胎蛋白抗体
hz-1673hz AEV(Avian Encephalomyelitis virus)  禽脑脊髓炎病毒AEV抗体
hz-4236R ADAM17/CD156b  肿瘤坏死因子α转换酶
hz-4710R PGT/Slco2a1  溶质载体蛋白家族21成员2抗体
hz-3574R ADAM10/MADM/CD156c  去整合素样金属蛋白酶10抗体
hz-4714R Salmonella  沙门氏菌抗体(多型)
hz-5846R ADAM11  去整合素样金属蛋白酶11抗体
hz-4715R Histone H3 (Tri Methyl K4)  **基化组蛋白H3抗体
hz-4718R MAGEA11  黑色素瘤相关抗原11抗体
hz-1385R ADAMTS12  整合素样金属蛋白酶与凝血酶1型-12抗体

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