PGL3-Basic Vector

合集下载

质粒测序引物

pDEST26
TREfor/CMV-Profor
T7/M13for
pDisplay
T7/CMV-Profor
c-mycrev
pDNR-LIB(MCS_A)
M13for,T7
pSG5-3,M13R
pDonar
M13F
M13R
pDONR201
PDONR-F
PDONR-R
pDONR207
PDONR-F
PDONR-R
T7,CMV-Profor
BGH rev,c-mycrev
pcDNA4/TO
TREfor/CMV-Profor
BGH rev
pcDNA4/V5-His(_A,_B,_C)
CMV-Profor,T7
BGH rev,V5rev
pcDNA4-TET/ON/AMP+
CMV-Profor
pcDNA5/FRT
T7/CMV-Profor
T7-Terminator
pET-20b(+)
T7
T7-Terminator
pET-21(-a,-b,-c,-d)(+)
T7
T7-Terminator
pET-22b(+)
T7
T7-Terminator
pET-23(-a,-b,-c,-d)(+)
T7
T7-Terminator
pET-24(-a,-b,-c,-d)(+)
pBR322(BamHI-site)
PBRforBam
PBRrevBam
pBR322(EcoRI/HindIII-sites)
pBRforEco
pBRrevHind
pBR325(BamHI-site)

同型半胱氨酸对FABP4启动子活性的影响

第28卷第29期中国现代医学杂志 Vol. 28 No.29 2018年10月 China Journal of Modern Medicine Oct. 2018收稿日期：2018-05-15*基金项目：国家自然科学基金（No ：81560084）；宁夏自然科学基金（No ：NZ16063）；宁夏高等学校科学研究项目（No ：NGY2016087）[通信作者] 姜怡邓，E-mail ：nyxxgjyd@DOI: 10.3969/j.issn.1005-8982.2018.29.005文章编号： 1005-8982（2018）29-0025-06同型半胱氨酸对FABP 4启动子活性的影响*熊建团1，李旭生2，杨安宁3，杨松昊3，邓梅3，王磊3，高源3，李南1，杨晓玲3，贾月霞3，姜怡邓3（1.宁夏医科大学药学院，宁夏银川 750004；2.宁夏医科大学总医院骨科，宁夏银川750004；3.宁夏医科大学基础医学院，宁夏银川 750004）摘要：目的通过克隆脂肪酸结合蛋白4（FABP4）基因启动子，确定其活性核心区和功能片段，分析心血管疾病独立危险因子同型半胱氨酸（Hcy）对FABP 4启动子活性的影响。

方法应用生物信息学预测FABP 4基因启动子区顺式转录作用元件和反式作用因子，以pGL3-Basic 为载体，采用基因重组法构建启动子截取片段，转染HEK-293A 细胞，观察不同截取片段的荧光素酶活性变化，确定活性最强的片段。

进一步将核心启动子片段（-2000/-1）转染巨噬细胞，观察不同浓度Hcy 和DNA 甲基化抑制剂5-氮杂胞苷（AZC）对FABP 4基因启动子活性的影响。

结果不同长度截取片段转染HEK-293A 细胞后检测荧光素酶活性结果显示，与pGL3对照组比较，-2000/-1片段转录活性最强。

将核心启动子片段（-2000/-1）转染巨噬细胞并用不同浓度Hcy 干预后，与0μmol/L Hcy 组比较，100和200μmol/L Hcy 组启动活性升高；与100μmol/L Hcy 组比较，在100μmol/L Hcy 基础上用AZC 干预后，FABP 4基因启动活性升高（P <0.05）。

报告基因和荧光淬灭

优缺点
每个FRET以上列出的方法都有长处和短处。例如，一方面，两个通道成像法是最简单，但需要最复杂的控件集。另一方面，FLIM的可以得到明确的测量FRET 效率，但是昂贵。
Principle of the assay
Ryoichi Arai et al. Protein Eng. 2000;13:369-376
FRET技术在细胞生物学应用
敏排放 -双通道成像使用一种算法，纠正串扰激发和发射受体漂白 -也被称为施主去猝灭受体光漂白时，这种技术措施增加了供体发射荧光寿命成像显微镜（FLIM） -荧光蛋白（或其他荧光）体寿命测量的变化光谱成像 -令人兴奋的一个或两个波长，测量供体和受体的完整光谱剖面荧光偏振成像 -测量偏振平行和垂直于激发高信号噪声
FRET
青色荧光蛋白（cyan fluorescent protein, CFP）、黄色荧光蛋白（yellow fluorescent protein, YFP）为目前蛋白-蛋白相互作用研究中最广泛应用的 FRET对。CFP的发射光谱与YFP的吸收光谱相重叠。将供体蛋白CFG和受体蛋白YFG分别与两种目的蛋白融合表达。当两个融合蛋白之间的距离在5-10nm的范围内，则供体CFP发出的荧光可被YFP吸收，并激发YFP发出黄色荧光，此时通过测量CFP荧光强度的损失量来确定这两个蛋白是否相互作用。两个蛋白距离越近，CFP所发出的荧光被YFP接收的量就越多，检测器所接收到的荧光就越少。如Tsien & Miyawaki采用FRET技术检测细胞内Ca2+的浓度变化。他们将CFP 和YFP分别于钙调蛋白和钙调蛋白结合肽融合表达于同一个细胞内。当细胞内具有高的Ca2+浓度时，钙调蛋白和钙调蛋白结合肽结合，可诱发FRET，使受体蛋白YFP发出黄色荧光，因此细胞呈黄色。当细胞内Ca2+浓度低时，FRET几乎不发生，因此检测时CFP被激发，发出绿色荧光，细胞呈绿色。

质粒图谱查询方法

3.google scholar: / 有些质粒是经过改造的，所以通过上述方法不能查询到相应信息。这时，可以在google scholar中输入质粒名称，可以直观地看哪些学者在何文章中使用了该质粒，从而可了解到质粒的来源;或者籍此向作者咨询或索取。 4.尝试从各大生物公司，例如invitrogen网站查询. 5. 这个网站收录了大量图谱： http://www.embl-hamburg.de/~geerlof/webPP/vectordb/bact_vectors/table.html
Pe
te
rX u
file:///D|/中科院/Selective Serotonin Transporter/质粒信息/质粒图谱查询方法.txt
file:///D|/中科院/Selective Serotonin Transporter/质粒信息/质粒图谱查询方法.txt（第 2／6 页）[2011/8/4 18:39:52]
By
0099--pGE-1—Stratagene--RNAi载体 0100--pSUPER.p53—OligoEngine--RNAi载体 0101--palter-ex1--promega 0102--pACYCDuet-1--NOVAGEN 0103--pEX lox(+) Vector—NOVAGEN--原核表达 0104--质粒名称：pBACgus-8 Transfer Plasmid—NOVAGEN--CHUANSUO 0105--pSCREEN?-1b(+) Vector Map—novagen--筛选 0106--PGEX-2T--BD Co--pDsRed2--Clontech 0107--pbgal-Basic—Clontech--mammalian reporter vector 0108—pBI—Clontech--express two genes of interest from a bidirectional tet-responsive promoter 0109--质粒名称：pbgal-Control—Clontech--mammalian reporter vector 0110-- pGEX-5X-1--原核表达 0111--pBI-EGFP—Clontech--pBI-EGFP-- coexpress 0112--pBI-G—Clontech--pBI-G--express b-galactosidase 0113--pBI-GL—Clontech--pBI-GL --express luciferase and b-galactosidase 0114--pCMS-EGFP—Clontech--mammalian expression vector 0115--pd2EYFP-1—Clontech--启动子测定 0116--质粒名称--pd2EYFP-N1—Clontech--融合表达 0117--pd4EGFP-Bid—Clontech--融合表达 Bid 0118--pDNR-CMV—Clontech--pDNR-CMV 0119--pDNR-EGFP Vector—Clontech 0120--pDNR-LacZ –Clontech 0121--pECFP-Endo—Clontech--真核表达0122--pECFP-ER—Clontech--真核表达0123--pEGFP-Actin—Clontech--真核表达0124--pGAD GH--Clontech--酵母表达 0125--pGADT7-Rec –Clontech--酵母表达 0126--pGADT7-RecAB—Clontech--酵母表达 0127--pGADT7-Rec2—Clontech--酵母表达 0128--pGBKT7—Clontech--酵母表达 0129--pHAT 10/11/12—Clontech 0130--pHAT20—Clontech 0131—pHygEGFP—Clontech 0132—pLacZi—Clontech 0133—pM—Clontech--pM is used to generate a fusion of the GAL4 DNA-BD 0134--pPKCa-EGFP—Clontech 0135--pPKCb-EGFP—Clontec 0136--pSIREN-DNR Vector—Clontech--RNAi 0137--pSIREN-DNR-DsRed-Express Vector—Clontech--RNAi 0138--pSIREN-RetroQ—Clontech--RNAi 0139--pIRES-EYFP—Clontech--RNAi 0140--pSRE-Luc—Clontech--RNAi 0141--pTK-neo—novagen--原核表达 0142--pZsGreen Vector—Clontech--pZsGreen is a pUC19-derived prokaryotic expression vector 0143--pTandem-1—novagen--原核表达 0144--pZsGreen1-C1Vector—Clontech----真核表达 0145--质粒名称：M13mp18—novagen--原核表达 0146--pZsGreen1-DR Vector—Clontech--真核表达 0147--PZsGreen1-N1 Vector—Clontech --真核表达 0148--T7Select415-1b—novagen----真核表达 0149--pZsYellow Vector—Clontech --真核表达 0150—pTimer—Clontech --真核表达 0151--pTA-Luc—Clontech --真核表达 0152--pTAL-Luc—Clontech --真核表达 0153--pTA-SEAP—Clontech --真核表达 0154--pTAL-SEAP—Clontech --真核表达 0155--pTet-On—Clontech --真核表达 0156--pTet-Off—Clontech --真核表达 0157--pTet-ATF—Clontech --真核表达 0158--pTet-CREB—Clontech --真核表达

pGL3-Basic_Vector质粒图谱及其说明

Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA·Fax 608-277-2516 ·1.Description ..........................................................................................................22.Product Components and Storage Conditions ............................................23.pGL3 Vector Maps and Sequence Reference Points . (2)A.pGL3-Basic Vector................................................................................................3B.pGL3-Enhancer Vector........................................................................................4C.pGL3-Promoter Vector........................................................................................5D.pGL3-Control Vector...........................................................................................64.Cloning Methods .. (7)A.Cloning Strategies.................................................................................................7B.Preparation of pGL3 Vectors and Insert DNA for Cloning...........................8C.Transformation Protocols for pGL3 Vectors....................................................8D.Isolation of Plasmid DNA (8)5.Transfection of Mammalian Cells ..................................................................96.Assay of Luciferase Activity ............................................................................97.Sequencing of Luciferase Reporter Vectors .. (11)8.Appendix mon Structural Elements of the pGL3 LuciferaseReporter Vectors ................................................................................................12B.Advantages of the pGL3 Vectors.....................................................................13C.The pGL3 Vectors luc + Gene............................................................................14D.Mapping Genetic Elements Located Within DNA position of Buffers and Solutions............................................................16F.References............................................................................................................17G.pGL3-Basic Vector Restriction Sites.................................................................18H.pGL3-Enhancer Vector Restriction Sites.........................................................20I.pGL3-Promoter Vector Restriction Sites.........................................................23J.pGL3-Control Vector Restriction Sites............................................................26K.Related Products. (28)pGL3 Luciferase Reporter VectorsAll technical literature is available on the Internet at: /tbs/ Please visit the web site to verify that you are using the most current version of this Technical Bulletin. Please contact Promega Technical Services if you have questions on useofthissystem.E-mail:********************1.DescriptionThe pGL3 Luciferase Reporter Vectors(a–c)provide a basis for the quantitativeanalysis of factors that potentially regulate mammalian gene expression. Thesefactors may be cis-acting, such as promoters and enhancers, or trans-acting,such as various DNA-binding factors. The backbone of the pGL3 LuciferaseReporter Vectors is designed for increased expression, and contains a modifiedcoding region for firefly (Photinus pyralis) luciferase that has been optimized formonitoring transcriptional activity in transfected eukaryotic cells. The assay ofthis genetic reporter is rapid, sensitive and quantitative. In addition, theseLuciferase Reporter Vectors contain numerous features aiding in the structuralcharacterization of the putative regulatory sequences under investigation.2.Product Components and Storage ConditionsProduct Size Cat.# pGL3-Control Vector20μg E1741 pGL3-Basic Vector20μg E1751 pGL3-Promoter Vector20μg E1761 pGL3-Enhancer Vector20μg E1771 Information on related products, including the Luciferase Assay System, is provided inSections 4–7 and 8.K.Storage Conditions:Store the pGL3 Luciferase Reporter Vectors at –20°C.3.pGL3 Vector Maps and Sequence Reference PointsThe listings of restriction sites for the pGL3 Luciferase Reporter Vectors areprovided in Section VIII.G–J.Note: The specific transcriptional characteristics of the pGL3 Vectors willvary for different cell types. This may be particularly true for COS cells,which contain the SV40 large T antigen. The SV40 large T antigen promotesreplication from the SV40 origin, which is found in the promoter of thepGL3-Promoter and pGL3-Control Vectors. The combination of large T antigenand SV40 origin will result in a higher copy number of these vectors in COScells, which in turn may result in increased expression of the reporter genecompared to other cell and vector combinations.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA·3.A. pGL3-Basic VectorThe pGL3-Basic Vector lacks eukaryotic promoter and enhancer sequences,allowing maximum flexibility in cloning putative regulatory sequences.Expression of luciferase activity in cells transfected with this plasmid depends on insertion and proper orientation of a functional promoter upstream from luc +. Potential enhancer elements can also be inserted upstream of the promoter or in the BamHI or SalI sites downstream of the luc + gene.Figure 1. pGL3-Basic Vector circle map. Additional description: luc +, cDNAencoding the modified firefly luciferase; Amp r , gene conferring ampicillin resistance in E. coli ; f1 ori, origin of replication derived from filamentous phage; ori, origin of replication in E. coli.Arrows within luc + and the Amp r gene indicate the direction of transcription; the arrow in the f1 ori indicates the direction of ssDNA strand synthesis.pGL3-Basic Vector Sequence Reference Points:Promoter (none)Enhancer(none)Multiple cloning region 1–58Luciferase gene (luc +)88–1740GLprimer2 binding site 89–111SV40 late poly(A) signal 1772–1993RVprimer4 binding site2080–2061ColE1-derived plasmid replication origin 2318β-lactamase gene (Amp r )3080–3940f1 origin4072–4527upstream poly(A) signal 4658–4811RVprimer3 binding site4760–4779Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA·Fax 608-277-2516 ·(for 0746V A 08_4AThe pGL3-Enhancer Vector contains an SV40 enhancer located downstream of luc + and the poly(A) signal. This aids in the verification of functional promoter elements because the presence of an enhancer will often result in transcription of luc + at higher levels.Figure 2. The pGL3-Enhancer Vector circle map. Additional description: luc +,cDNA encoding the modified firefly luciferase; Amp r , gene conferring ampicillin resistance in E. coli ; f1 ori, origin of replication derived from filamentous phage; ori,origin of plasmid replication in E. coli . Arrows within luc + and the Amp r gene indicate the direction of transcription; the arrow in f1 ori indicates the direction of ssDNA strand synthesis.pGL3-Enhancer Vector Sequence Reference Points:Promoter(none)Multiple cloning region 1–58Luciferase gene (luc +)88–1740GLprimer2 binding site 89–111SV40 late poly(A) signal 1772–1993Enhancer2013–2249RVprimer4 binding site2307–2326ColE1-derived plasmid replication origin 2564β-lactamase gene (Amp r )3329–4186f1 origin4318–4773upstream poly(A) signal 4904–5057RVprimer3 binding site5006–5025Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA·0745V A 08_4AThe pGL3-Promoter Vector contains an SV40 promoter upstream of theluciferase gene. DNA fragments containing putative enhancer elements can be inserted either upstream or downstream of the promoter-luc + transcriptional unit.Figure 3. The pGL3-Promoter Vector circle map. Additional description: luc +,cDNA encoding the modified firefly luciferase; Amp r , gene conferring ampicillin resistance in E. coli ; f1 ori, origin of replication derived from filamentous phage; ori,origin of plasmid replication in E. coli . Arrows within luc + and the Amp r gene indicate the direction of transcription; the arrow in f1 ori indicates the direction of ssDNA strand synthesis.pGL3-Promoter Vector Sequence Reference Points:Enhancer(none)Multiple cloning region 1–41Promoter48–250GLprimer2 binding region 281–303Luciferase gene (luc +)280–1932SV40 late poly(A) signal 1964–2185RVprimer4 binding region2253–2272ColE1-derived plasmid replication origin 2510β-lactamase gene (Amp r )3272–4132f1 origin4264–4719Upstream poly(A) signal 4850–5003RVprimer3 binding region4952–4971Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA·Fax 608-277-2516 ·51115212832360748V A 08_4AThe pGL3-Control Vector contains SV40 promoter and enhancer sequences,resulting in strong expression of luc + in many types of mammalian cells. This plasmid is useful in monitoring transfection efficiency, in general, and is a convenient internal standard for promoter and enhancer activities expressed by pGL3 recombinants.Figure 4. pGL3-Control Vector circle map. Additional description: luc +, cDNA encoding the modified firefly luciferase; Amp r , gene conferring ampicillin resistance in E. coli ; f1 ori, origin of replication derived from filamentous phage; ori, origin of plasmid replication in E. coli . Arrows within luc + and the Amp r gene indicate the direction of transcription; the arrow in f1 ori indicates the direction of ssDNA strand synthesis.pGL3-Control Vector Sequence Reference Points:Multiple cloning region 1–41Promoter48–250Luciferase gene (luc +)280–1932GLprimer2 binding site 281–303SV40 late poly(A) signal 1964–2185Enhancer2205–2441RVprimer4 binding site2499–2518ColE1-derived plasmid replication origin 2756β-lactamase gene (Amp r )3518–4378f1 origin4510–4965upstream poly(A) signal 5096–5249RVprimer3 binding site5198–5217Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA·(for 0747V A 08_4AFigure 5. pGL3 Vector multiple cloning regions. Shown are the upstream and downstream cloning sites and the locations of the sequencing primers (GLprimer2,RVprimer3 and RVprimer4). The large primer arrows indicate the direction ofsequencing. The positions of the promoter (in the pGL3-Promoter and pGL3-Control Vectors) and the enhancer (in the pGL3-Enhancer and pGL3-Control Vectors) are shown as insertions into the sequence of the pGL3-Basic Vector. (Note that the promoter replaces four bases [AAGT] of the pGL3-Basic Vector.) The sequence shown is of the DNA strand generated from the f1 ori.4.Cloning Methods4.A. Cloning StrategiesThe restriction sites for XhoI and SalI have compatible ends, as do BglII and BamHI. Therefore, cloning into the XhoI or BglII sites upstream of luc +, or the downstream SalI or BamHI sites, allows easy interchange of DNA insertsbetween upstream and downstream positions relative to the luciferase reporter gene. Thus, positional effects of a putative genetic element may be readily tested. Cloning fragments into a single site will generally yield both possible orientations relative to the reporter gene, making these effects also readily testable.The other upstream restriction sites may be used for cloning. However, note that some of the sites are required for generating nested deletions (see Section 8.D). Specifically, the KpnI or SacI site is needed to generate a 3´ overhang upstream of the insert.Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA·Fax 608-277-2516 ·CATTCCGGTACTGTTGGTAAAGCCACCATGGAAGACGCCAAAAACATAAAG . . . (1892bp) . . . GGATCCGTCGACRVprimer35′GGTACCGAGCTCTTACGCGTGCTAGCCCGGGCTCGAGATCTGCGATCTAAGTAAGCTTGG . . .KpnI Acc65ISacIMluINheIXmaI SmaIXhoIBglIIHindIIISV40Enhancerluc+ Coding Region ′BamHI SalI0756M A 08_4A4.B.Preparation of pGL3 Vectors and Insert DNA for CloningThe fragment and vector DNA should be digested with restriction enzymesthat will generate compatible ends for cloning. In some cases, the ends of theDNA fragment may require modification, either by using synthetic linkers, bya PCR amplification using primers containing sites for appropriate restrictionenzymes, or by filling in the restriction site overhangs. It may be advantageousto treat the vector DNA with calf intestinal alkaline phosphatase (CIAP; Cat.#M2825) or TSAP Thermosensitive Alkaline Phosphatase (Cat.# M9910) toremove 5´ phosphate groups, thus preventing reclosure of the vector on itselfwithout an insert. Sufficient DNA should be prepared to perform controlreactions for digestion, ligation and transformation steps.To ensure capture of the correct insert DNA, the desired restriction fragmentcan be purified by electrophoresis on an acrylamide or agarose gel and thenrecovered from the gel by one of several methods, such as using the Wizard®PCR Preps DNA Purification System Technical Bulletin#TB118. Alternatively,unfractionated restriction fragments can be cloned into the target plasmid, andthe desired recombinant then can be identified by gel electrophoresis ofplasmid DNA.Protocols for restriction digestion, alkaline phosphatase treatment, linkerligation and transformation of competent cells can be found in MolecularCloning, A Laboratory Manual(1).4.C. Transformation Protocols for pGL3 VectorsBecause the Luciferase Reporter Vectors are supplied as modified DNA, E. colihosts may be either restriction + or restriction –. The use of a rec A host such asJM109 is preferred because this prevents undesirable recombination betweenthe insert and the host chromosomal DNA. A strain that has an F´ episome isrequired for ssDNA production.Grow JM109 on minimal plates (M-9) supplemented with 1.0mM thiamine-HCl prior to preparation of competent cells and transformation. This selectsfor the presence of the F´ episome.4.D. Isolation of Plasmid DNAThe Wizard®Plus SV Minipreps DNA Purification System (Cat.# A1340,A1470) may be used for small-scale preparation of plasmid DNA for screeningclones. DNA suitable for transfection may be purified using the PureYield™Plasmid Midipreps System (Cat.# A2492, A2495).Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA·5.Transfection of Mammalian CellsTransfection of DNA into eukaryotic cells may be mediated by cationic lipid compounds (2), calcium phosphate (3,4), DEAE-dextran (3,5), or electroporation (4). Transfection systems based on cationic lipids (TransFast™ Transfection Reagent, Transfectam®Reagent and Tfx™ Reagents) and calcium phosphate (Profection®Mammalian Transfection System) are available from Promega. Formore information on these transfection reagents, please request the TransFast™Transfection Reagent Technical Bulletin(#TB260), the Transfectam®Reagent Technical Bulletin(#TB116), the Tfx™-Reagents Technical Bulletin(#TB216) or the ProFection®Mammalian Transfection System Technical Manual(#TM012). All of thesedocuments are available on our web site at: /tbs/6.Assay of Luciferase ActivityExperimental strategies using firefly luciferase may involve the analysis of afew samples per day or as many as several thousand samples per hour, and equipment used to measure luminescence may vary from inexpensive, single-sample luminometers to high-end CCD luminometers. To support this widerange of applications, we have developed three luciferase assays with different,but complementary, characteristics: Luciferase Assay System (Cat.# E1500),Bright-Glo™ Luciferase Assay System (Cat.# E2610), Steady-Glo®LuciferaseAssay System (Cat.# E2510), and ONE-Glo™ Luciferase Assay System (Cat.#E6110). Reagent choice depends on the relative importance of experimentalformat, assay sensitivity, and luminescence duration.Table 1. Characteristics of Promega Luciferase Assay Reagents.LuciferaseBright-Glo™Steady-Glo®Assay ONE-Glo™Reagent Reagent Reagent ReagentFormat NH or H NH or H NH NH or HProcess continuous batch bench scale batch orcontinuous Number of Steps1141Sensitivity highest lower higher highSignal Half-Life~30 minutes~5 hours~12 minutes~50 minutes Precision High High High HighestCell Lysis Time~2 minutes~5 minutes NA~3 minutesmaximum maximumNH = nonhomogeneous (first create a lysate); H = homogeneous; NA = not applicablePromega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA·Fax 608-277-2516 ·6.Assay of Luciferase Activity (continued)The Luciferase Assay System has long been the standard reagent for routine laboratory analysis. Before using this reagent, cells from which the luciferase is to be measured must be washed and lysed. This reagent was optimized for high sensitivity in nonhomogeneous, single-sample measurements. The Luciferase Assay System requires a luminometer fitted with injectors to efficiently measure luminescence in 96-well plates.The Bright-Glo™, Steady-Glo®and ONE-Glo™ Reagents were developed to perform assay reactions within multiwell plates and in the presence of complete cell culture medium: no cell preparation steps such as washing or lysing are required before the luminescence reaction is initiated. All of these are single-step reagents, requiring only addition of the reagent before measuring luminescence. This makes them ideal reagents for efficient and precise quantitation in 96-, 384- and 1536-well plates.The Bright-Glo™ and Steady-Glo®Reagents are complementary in their characteristics based on the inverse relationship between luminescence duration and assay sensitivity (6). Generally, as the half-life of the luminescence increases, assay sensitivity decreases. The Steady-Glo®Reagent provides long luminescence duration (changing only about 10% per hour); however, toachieve this long luminescence duration, the assay sensitivity must be reduced. This reagent was designed for experiments in which many microplates are processed as a batch.In contrast, the Bright-Glo™ Reagent provides high assay sensitivity with shorter luminescence duration (<10% decrease per 5 minutes). This reagent is designed for general research applications and for experiments using robotics for continuous sample processing. Furthermore, as a result of increased sample capacity, the Bright-Glo™ Reagent provides greater assay sensitivity than the Luciferase Assay Reagent in most applications (6).The ONE-Glo™ Reagent provides the ultimate performance for luciferase assays. It features a high-sensitivity assay with extended duration. TheONE-Glo™ Reagent also demonstrates more robust performance and provides reagent handling enhancements.The Luciferase Assay System, Bright-Glo™ Reagent, Steady-Glo®Reagent and ONE-Glo™ Reagent provide the highest standards in assay quantitation, sensitivity and convenience. Since these reagents are based on the same underlying design principles, different reagents can be used as experimental needs change. For more information, request the Luciferase Assay System Technical Bulletin#TB281, the Steady-Glo®Luciferase Assay System Technical Manual#TM051, the Bright-Glo™ Luciferase Assay System Technical Manual#TM052, or the ONE-Glo™ Luciferase Assay System Technical Manual#TM292.When studying promoter functionalities, it is often desirable to include asecond reporter (e.g., Renilla luciferase) as an internal control for normalization. Plasmids derived from pGL3 or pGL4 vectors can be co-transfected with Renilla luciferase vectors, such as phRL-TK, and assayed using the Dual-Luciferase®Reporter Assay System (Cat.# E1910) or the Dual-Glo™ Luciferase AssaySystem (Cat.# E2920).Table 2. Characteristics of Promega Dual-Luciferase Assays.Dual-Luciferase®Dual-Glo™Assay AssayFormat NH HProcess bench scale batchNumber of Steps52Sensitivity higher lowerSignal Half-Life—firefly~9 minutes~2 hoursSignal Half-Life—Renilla~2 minutes~2 hoursPrecision High HighCell Lysis Time~10 minutes~15 minutesmaximum maximumNH = nonhomogeneous (first create a lysate); H = homogeneous7.Sequencing of Luciferase Reporter VectorsYou may desire to sequence the DNA inserted into the Luciferase Reporter Vectors. Two examples of such applications are to determine the exact positionof generated deletions and to confirm production of a site-specific mutation.Three primers are available for sequencing the pGL3 Vectors: RVprimer3 (Reporter Vector Primer 3) for sequencing clockwise across the upstreamcloning sites, RVprimer4 for sequencing counterclockwise across the BamHIand SalI cloning sites downstream of luc+, and GLprimer2 for sequencing counterclockwise upstream of luc+.RVprimer35´-CTAGCAAAATAGGCTGTCCC-3´RVprimer45´-GACGATAGTCATGCCCCGCG-3´GLprimer25´-CTTTATGTTTTTGGCGTCTTCCA-3´RVprimer3 is especially useful for identifying positions of nested deletions.Note:All three primers can be used for dsDNA sequencing, but onlyRVprimer4 and GLprimer2 also may be used for ssDNA sequencing.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA·Fax 608-277-2516 ·8.Appendix8.A. Common Structural Elements of the pGL3 Luciferase Reporter VectorsExcept for the inclusion of promoters and enhancers, the four pGL3 Luciferase Reporter Vectors are structurally identical. Each plasmid’s distinguishing features are summarized in Section 3. The pGL3 Vectors each contain a high-copy-number prokaryotic origin of replication for maintenance in E. coli , an ampicillin-resistance gene for selection, and a filamentous phage origin of replication (f1 ori) for single-stranded DNA (ssDNA) production. Restriction sites for insertion of DNA fragments are located upstream and downstream of the luciferase gene. Two of the upstream sites (XhoI and BglII) yield cohesive ends compatible with the downstream sites (SalI and BamHI, respectively),allowing the interchange of the DNA insert for rapid analysis of positional effects.Figure 6. Comparison of luciferase activities expressed in HeLa cells transfected with the pGL2-Control and pGL3-Control Reporter Vectors. The expression level of luc + is dramatically higher with the pGL3-Control Vectors. In repeatedexperiments with several cell lines, we observed 20- to 100-fold higher luciferase activity from cells transfected with pGL3-Control. Luciferase activity was measured with a Turner Designs luminometer. (Absolute light values and relative expressionprofiles may vary between different cell types.)1,4001,2001,0008006004002000Improved Expression Level with the pGL3-Control VectorConstruct TransfectedpGL3-ControlVectorpGL2-ControlVectorA v e r a g e R e l a t i v e L i g h t U n i t sFigure 7. A representative experiment comparing luciferase activities expressed in HeLa cells transfected with the pGL2 and pGL3 Vector series. The increase in luciferase expression observed with these new vectors provides greater sensitivity,while maintaining relatively low background luciferase expression.8.B. Advantages of the pGL3 VectorsThe pGL3 Reporter Vectors contain a modified firefly luciferase cDNAdesignated luc + and a redesigned vector backbone. These changes were made to increase luciferase expression, improve in vivo vector stability, and provide greater flexibility in performing genetic manipulations. The modified reporter vectors have resulted in luciferase expression levels dramatically higher than those obtained with pGL2 Reporter Vectors (Figure 6), while maintaining relatively low background luciferase expression (Figure 7).The substantial increase in the expression of luciferase observed with the pGL3Vectors provides greater sensitivity. It may now be possible to obtainmeasurable luciferase expression in cell types that are difficult to transfect or when studying weak promoter elements. Users of the pGL2 and pGL3 Vectors should be aware, however, that absolute light unit values and relative expression profiles vary between different cell types (7). Therefore, it is important to include the appropriate control vectors in all experiments.Further refinements have been made since the pGL3 Vectors became available.Our newest series of luciferase reporter vectors, the pGL4 Luciferase Vectors,provide additional features and benefits as compared to the pGL3 Vectors. For more information, see the pGL4 Luciferase Reporter Vectors Technical Manual #TM259 available at:/tbs/Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA·Fax 608-277-2516 ·80604020Construct TransfectedControl Basic Enhancer PromoterA v e r a g e R e l a t i v e L i g h t U n i t sA v e r a g e R e l a t i v e L i g h t U n i t sConstruct TransfectedControl Basic Enhancer Promoter 08.C. The pGL3 Vectors luc+ GeneModifications that distinguish the luc+ gene from the native luciferase genegenerally fall into four categories: i) the C-terminal tripeptide has beenremoved to eliminate peroxisome targeting of the expressed protein; ii) codon usage was improved for expression in plant and animal cells; iii) two potential sites of N-glycosylation were removed; and iv) several DNA sequence changes were made to disrupt extended palindromes, remove internal restriction sites, and eliminate consensus sequences recognized by genetic regulatory binding proteins, thus helping to ensure that the reporter gene itself is unaffected by spurious host transcriptional signals. (For a detailed description of themodifications to the luc+ gene, see reference 8.)Four major modifications were made to the vector backbone: i) the SV40 early poly(A) signal has been replaced with the SV40 late poly(A) signal to increase the efficiency of transcription termination and polyadenylation of theluciferase transcripts (9); ii) a synthetic poly(A) and transcriptional pause site (10,11) have been placed upstream of the multiple cloning site to terminatespurious transcription, which may initiate within the vector backbone; iii) the small T intron has been removed to prevent reduced reporter gene expression due to cryptic RNA splicing (12,13); and iv) a Kozak consensus sequence (14) has been inserted to increase the efficiency of translation initiation of theluciferase gene (7; Table 3).There is a newer luciferase gene available, luc2. The luc2gene not only shares the same features as luc+, but the sequence was codon-optimized forexpression in mammalian cells. For further information about the luc2genepresent in the pGL4 Luciferase Vectors, see Technical Manual #TM259available at: /tbs/Table 3. Changes Made to the pGL3 Vectors.Changes Made Purpose of Modification Reference Modifications made to Changes eliminate peroxisome(8)the luciferase gene targeting of expressed protein,(luc to luc+).eliminate consensus bindingsequences for various geneticregulatory proteins, improvecodon usage for mammalianand plant cells, and provideconvenient restriction sites.A unique NcoI site created Ability to create N-terminalat 5´ end of luc+ gene. NcoI gene fusions with luc+sites removed from SV40 using unique NcoI site.enhancer and promoterregions.Intron from SV40 small Intron from SV40 small T (12,13)T antigen removed. antigen can reduceexpression when placed 3´of certain genes due tocryptic splicing.Poly(A) site for back-Avoids possible recombination (9,10)ground reduction changed between two SV40 poly(A)from SV40 early site to a sequences in thesynthetic poly(A) and same plasmid.transcriptional pause site.Poly(A) signal for luc+ Late SV40 poly(A) signal is(7)changed from early to more efficient than early SV40late SV40 poly(A) signal.poly(A).Kozak consensus Provides optimal (14) sequence created translation efficiency.immediately 5´ of theluc+ gene.Unique XbaI site User convenience; facilitatescreated just downstream subcloning of the luc+ gene.of the luc+ gene.SmaI site moved to User convenience; blunt-ended insertsinternal position in can now be cleaved on either sidemultiple cloning region.by restriction endonucleases.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA·Fax 608-277-2516 ·。

pgl3-basic vector中文

荧光素酶报告基因载体 —— pGL3产品包装目录号价格pGL3-Control Vector 20ug E1741pGL3-Basic Vector 20ug E1751pGL3-Promoter Vector 20ug E1761pGL3-Enhancer Vector 20ug E1771描述: 荧光素酶报告基因载体系列pGL3，为定量具有调节哺乳动物基因表达潜在能力的各种因子的活性提供一个分析基础。

这些可以是顺式或反式作用因子。

经重新设计，pGL2荧光素酶报告基因载体系列的骨架成为了pGL3系列载体的骨架，提高了表达量。

重新设计包括对萤火虫(Photinus pyralis)荧光素酶的编码区进行修饰，使其在被转染的真核细胞中能进行最优转录活性监控。

这个遗传报告基因的检测快速，灵敏，并且定量准确。

另外，荧光素酶报告基因载体系列具有各种不同特性，以帮助了解所研究的假设调节序列的结构特征。

特点：●易于使用：位于luc+ 基因5'末端的Nco I 位点允许使用这个唯一的Nco I位点制造与报告基因融合的蛋白。

●灵活：可从位于多克隆位点区的Sma I 位点插入平端片段于克隆位点区，并被其它限制性内切酶从两边切下。

●多功能：在紧靠luc+ 基因下游创造的Xba I 位点可促进mRNA 3'不翻译区的插入或亚克隆荧光素酶基因。

操作手册：技术手册： TM033保存条件：-20℃，菌株保存在-70℃。

GenBank®/EMBL 提取号码：pGL3-Basic: U47295. pGL3-Control: U47296. pGL3-Enhancer: U47297. pGL3-Promoter: U47298.注意：载体pGL3的特定转录特征随细胞类型的不同而不同。

特别是对于含有SV40大T抗原的COS 细胞，更是如此。

SV40大T抗原促进从SV40复制起点的复制，这个复制起点位于pGL3-Promoter和pGL3-Control载体的启动子上。

8 常用载体通用引物

pBudCE4.1(MCSII)
EF-1α Fwd
BGH
pBudCE4.1/lacZ/CAT
CMV-Profor,T7
c-mycrev,EBVrev
pBV220
pBV220F
PBV220R
pCAL-c
T7/Tac-Profor
T7-Terminator
pCAL-n
T7/Tac-Profor
T7-Terminator
M13rev/Tac-Profor
pcDNA3
T7/CMV-Profor
BGH rev/SP6
pcDNA3.1(+/-)
pcDNA3.1F/T7/CMV-Profor
BGH rev
pcDNA3.1/His(_A,_B,_C)
T7/CMV-Profor
BGH rev
pcDNA3.1/myc-His(_A,_B,_C)
BAC1
BAC2
pBAD
pBAD-F
pBAD-R
pBAD/HisABC
pBAD-5'
pBBR1MCS
T3/M13rev
T7/M13for
pBD-GAL4
pBD-GAL4-F
PBD-GAL4-R
pBI121
35S
NOS
pBIND
GAL4-Bdfor
T3,EBVrev
pBK-CMV
M13F/T7
M13R/T3
载体名称
上游引物（5'primer）
下游引物（3'primer）
P3*FLAG-CMV
CMV-30(825-854)
CMV-24(1129-1152)
pAc5.1/V5-His(_A,_B,_C)

载体——精选推荐

NEB
原核表达载体
NEB
配套大肠杆菌
Amersham 原核表达载体
Amersham 原核表达载体
Amersham 原核表达载体
Invitrogen 原核表达载体
Lucigen
原核表达菌株
Stratagene 原核表达菌株
Qiagen
原核表达载体
Qiagen
原核表达载体
Invitrogen 基因载体
700 元现货 800 元现货
900 元现货
800 元现货 800 元现货 800 元现货 800 元现货 800 元现货 700 元现货 800 元现货 800 元现货 800 元现货 800 元现货 900 元现货 1000 元现货 1000 元现货 1000 元现货 900 元现货 1000 元现货 1500 元现货 600 元现货 300 元现货 300 元现货 600 元现货 600 元现货 800 元现货 900 元现货 900 元现货 900 元现货
BL21(DE3) BL21(DE3)plysS Rosetta(DE3) Rosetta(DE3)plysS Rosetta-gami(DE3) Rosetta-gami B(DE3)pLysS Orgami(DE3) OrgamiB(DE3) BLR(DE3) pTWIN1 pTYB11 pMXB10 pTXB1 ER2566 pGEX-6P-1 pGEX-4T-2 pGEX-4T-3 pRset-a C41(DE3) BL21 codonplus（DE3）RIPL TAGZyme pQE-2 pQE-Trisystem pOG44 pORF-lacZ pORF-HSV1tk pSP73 pUC18 pUC19 pBR322 pSP64 pBluescript II SK(+) SuperCos 1 pGAPZα A pPICZα A

载体名称

pBudCE4.1(MCSII)
EF-1α Fwd
BGH
pBudCE4.1/lacZ/CAT
CMV-Profor,T7
c-mycrev,EBVrev
pBV220
pBV220F
PBV220R
pCAL-c
T7/Tac-Profor
T7-Terminator
pCAL-n
T7/Tac-Profor
T7-Terminator
pBR322(BamHI-site)
PBRforBam
PBRrevBam
pBR322(EcoRI/HindIII-sites)
pBRforEco
pBRrevHind
pBR325(BamHI-site)
PBRforBam
PBRrevBam
pBR329(BamHI-site)
PBRforBam
PBRrevBam
EBVrev
pCI-neo
T7（17BASE）
EBVrev,T3
pCMS-EG7
EBVrev,T3
pCMV-3Tag-4A
T3 /PFLAG-CMV-F
T7
pCMV5
pCMV5F
PCMV5R
pCMV6
CMV-Profor
pCMV6-3、HGHrev
pCMV6-XL4
CMV-Profor/T7
pcDNA3.3TOPOTA
CMV-Profor
TKPAreverse
pcDNA4/HisMax(_A,_B,_C)
Xpressfor
BGH rev
pcDNA4/HisMax-TOPO
Xpressfor
BGH rev
pcDNA4/HisMax-TOPO/lacZ

荧光素酶实验具体步骤及说明

双荧光素酶实验具体步骤及说明设计理念在检测一个报告基因测量结果的变化时，实验的准确性常被一些客观实验因素影响，如：细胞数目不均一、细胞代次不统一、转染效率不一致等等。

引入一个内参报告基因，以此来标定检测用报告基因的测量结果，从而达到有效地减少实验误差的目的。

系统工作原理双荧光素酶报告系统包括两个报告基因，一个检测用报告基因A 与一个内参用报告基因B。

一般是将检测用报告基因A 序列与调控启动子序列偶连在一起，或将报告基因A 序列与待检序列串联，报告基因A 荧光强度的相对改变与偶联的调控启动子的活性或待检序列的改变相关。

内参用报告基因B 与一个组成型启动子偶联，这个启动子不受各种实验条件变化的干扰，所以报告基因B 的荧光为组成型表达，可以作为内参。

检测用报告基因A、内参用报告基因B 可以装在不同的载体上，共同转染细胞，通过这种方法，把可能削弱实验准确性的内在因素的变化降到最小。

有的载体上同时含有检测用报告基因A 和内参用报告基因B，这样使实验操作更加准确、简便、快捷。

常用载体（Promega ）实验用报告基因：pGL3 Luciferase Reporter Vectors （pGL3-Control Vector 、pGL3-Basic Vector 、pGL3-Promoter Vector、pGL3-Enhancer Vector）；psiCHECK TM Vector（psiCHECK TM -1 Vector、psiCHECK TM -2 Vector*）；pmirGLOVector*。

内参用报告基因（组成型表达海肾荧光素酶）：pRL-TK；pRL-SV40；pRL-CMV；pRL-Null。

注：psiCHECK TM -2 Vector*、pmirGLO Vector* 同时表达萤火虫荧光素酶（Firefly Lucifera se ）及海肾荧光素酶（Renilla Lucifera s e），故无需准备对照pRL 系列内参质粒，可直接进行下游双荧光素酶检测实验。

GA引物载体对照表1

AmpR AmpR
AmpR ZeocinR ZeocinR ZeocinR ZeocinR ZeocinR ZeocinR
KanaR
pGEM-1 pGEM-11z pGEM-3 pGEM-3z pGEM-3Zf(+)(-) pGEM-4 pGEM-4Z pGEM-4z pGEM-5z pGEM-5Zf(+)(-) pGEM-7z pGEM-7Zf(+)(-) pGEM-9z pGEM-9Zf(+)(-) pGEM-T pGEM-T pGEM-T_Easy pGEM-Teasy pGEX-4T-1(-2,-3) pGEX-5X-1(-2,-3) pGEX-6P-1(-2,-3) pGL2 pGL2-Basic pGL3(MCSI,luc+upstream) pGL3(MCSII,luc+downstream) pGL3-Basic(MCSI) pGL3-Basic(MCSII) pGL4(MCSI,luc+upstream) pGL4(MCSII,luc+downstream) pIRES(MCSI) pIRES(MCSII) pIRESneo2 pLVX-IRES-ZsGreen1 pMALc PMAL-C2X pMALd pMALe pMB438 Pmd18/19-T pPIC3.5K pPIC6A pPIC6alphaA pPIC6alphaB pPIC6alphaC
AmpR&KanaR AmpR&KanaR AmpR AmpR AmpR&KanaR AmpR&KanaR AmpR AmpR&KanaR
pET-3(-a,-b,-c,-d) pET-30_Ek/LIC pET-30_Xa/LIC pET-30a(-b,-c)(+) pET-31b(+) pET-32a(-b,-c)(+) pET-33b(+) pET-34b(+) pET-35b(+) pET-36b(+) pET-37b(+) pET-38b(+) pET-39b(+) pET-40b(+) pET-41a(-b,-c)(+) pET-42a(-b,-c)(+) pET-44a(-b,-c)(+) pET-44a_EK/LIC pET-45b(+) pET-46_Ek/LIC pET-5(-a,-b,-c) pET-7 pET-9(-a,-b,-c,-d) pETDuet-1(MCSI) pETDuet-1(MCSII) pFastBac-Dual pFastBacDual(MCSI,PHpromoter) pFastBacDual(MCSII,p10promoter) pFastBac-GT pFastBac-HT PFLAG-CMV4 pGADT7 pGADT7 pGAD-T7 pGAPZA pGAPZalphaA pGAPZalphaB pGAPZalphaC pGAPZB pGAPZC pGBD-T7 pGBKT7 pGBKT7

质粒命名规则

质粒命名规则一、概述质粒是一种广泛应用于分子生物学研究中的载体，其命名规则对于实验室管理和数据交流具有重要意义。

本文将详细介绍质粒命名的规则。

二、基本要素1. 质粒名称应简洁明了，避免使用过长或过于复杂的名称。

2. 质粒名称应具有唯一性，不同质粒之间不能出现重复。

3. 质粒名称应具有信息量，能够反映出质粒的基本性质和功能特点。

三、命名方式1. 基于来源根据质粒来源进行命名，如pUC19（来源于E.coli）。

2. 基于功能根据质粒的功能进行命名，如pGEX-6P-1（用于表达融合蛋白）。

3. 基于结构根据质粒结构进行命名，如pBluescript II SK+（含有多个限制酶切位点）。

4. 基于序列根据质粒序列进行命名，如pET28a（含有T7启动子和His标签）。

四、命名元素1. 质粒类型使用常见缩写表示不同类型的质粒：p：常规质粒（plasmid）v：病毒载体（viral vector）b：大肠杆菌人工染色体（bacterial artificial chromosome）y：酵母人工染色体（yeast artificial chromosome）2. 来源使用缩写表示不同来源的质粒：pUC：来源于E.coli DH5αpBR：来源于E.coli K12pRS：来源于酵母菌3. 功能使用缩写表示不同功能的质粒：GEX：用于表达融合蛋白（glutathione S-transferase fusion expression）GST：含有谷胱甘肽S-转移酶标签（glutathione S-transferase tag）His：含有组氨酸标签（histidine tag）4. 结构使用缩写表示不同结构的质粒：KS：含有多个限制酶切位点（multiple cloning sites）SK+：含有T3、T7和SP6启动子（T3/T7/SP6 promoter）5. 序列使用缩写表示不同序列的质粒：ET28a：含有T7启动子和His标签（T7 promoter and His tag）HA-pcDNA3.1(+)：含有人流感病毒血凝素标签和CMV启动子（hemagglutinin tag and CMV promoter）五、示例命名1. 基于来源pUC19（来源于E.coli DH5α）pBR322（来源于E.coli K12）2. 基于功能pGEX-6P-1（用于表达融合蛋白）pcDNA3.1(+)-HA（含有人流感病毒血凝素标签）3. 基于结构pBluescript II SK+（含有多个限制酶切位点和启动子）pET28a（含有T7启动子和His标签）4. 基于序列pGL3-Basic（含有火梨酸激酶启动子和荧光素酶基因）pEGFP-N1（含有绿色荧光蛋白基因和CMV启动子）六、总结质粒命名规则是实验室管理和数据交流的重要组成部分，应根据质粒类型、来源、功能、结构和序列等要素进行命名，以便于实验室管理和数据交流。

鸡卵清蛋白启动子功能分析及验证

鸡卵清蛋白启动子功能分析及验证摘要转基因鸡输卵管生物反应器具有高产蛋能力，转基因鸡群快速扩繁能力和与人类相似的蛋白糖基化形式，在药用蛋白生产方面具有不可替代的作用。

到目前为止，相继建立了鸡蛋卵白（eggwhite）中表达干扰素、溶菌酶、抗体、促红细胞生成素、表皮生长因子等重组蛋白的转基因鸡。

但是对于不同长度卵清蛋白启动子（OVP）活性，以及雌激素应答元件（ERE）和卵清蛋白上游启动子转录因子（COUP-TF）调控作用缺乏系统研究；其次，作为外源基因转移的有效载体，鸡原始生殖细胞（PGCs）分离培养方法也待探讨。

本研究的目的是确定鸡OVP核心区域，筛选高转录活性的OVP; 探讨ERE和COUP-TF调控作用；摸索鸡PGCs适宜培养方法；通过体外和体内验证试验，确定可望用于制备输卵管生物反应器的OVP。

1. CRISPR-dCas9-VP64介导的鸡卵清蛋白启动子转录活性分析。

将启动子cERE-cOVP-2785、cOVP-2785分别亚克隆至pGL3-Basic载体，构建了荧光素酶表达载体pGL3-cERE-cOVP-2785、pGL3 -cOVP-2785。

利用Lipofectamine™ 2000将上述2个质粒以及pGL3cOVP-1548分别与pRL-TK质粒共转染293T细胞，检测荧光素酶活性，评估三个启动子的转录活性。

利用在线软件Optimized CRISPR Design，依据鸡卵清蛋白（OV A）基因第一内含子、转录起始位点上游区域DNA序列，共设计11对sgRNA；依据鸡ERE序列设计了2对sgRNA, 分别构建了11个pGL3-U6-OVPsgRNA 和2个pGL3-U6-EREsgRNA重组质粒；将pGL3-cERE-cOVP-2785、pcDNA-dCas9或pcDNA-dCas9-VP64质粒分别与单个或多个sgRNA表达质粒共转染293T细胞，检测荧光素酶活性，评估启动子和ERE对荧光素酶表达的影响。

PGC_1_辅助激活人PEPCK基因转录

第32卷第2期2011年3月中山大学学报（医学科学版）JOURNAL OF SUN YAT-SEN UNIVERSITY（MEDICAL SCIENCES）Vol．32No．2Mar．2011 PGC-1α辅助激活人PEPCK基因转录何圣清，陈燕铭*，王曼曼，舒冏，梁华，任琢琢，曾龙驿，翁建平（中山大学附属第三医院内分泌科，广东广州510630）摘要：【目的】构建人磷酸烯醇式丙酮酸羧激酶（PEPCK）基因启动子荧光素酶报告质粒PGL3-hPCK-luc，并探讨过氧化物酶体增殖物活化受体γ辅助激活因子1α（PGC-1α）在转录因子肝细胞核因子4α（HNF4α）介导下对人PEPCK基因启动子活性的影响。

【方法】从人全血基因组DNA中克隆PEPCK启动子基因片段，并重组进荧光素酶报告载体PGL3-basic，将pcDNA3．1-PGC-1α、pcDNA3．0-HNF4α表达质粒与PGL3-hPCK-luc按不同组合共转染进人肝癌细胞株HepG2细胞或正常人肝细胞株LO2细胞，培养48h后检测细胞裂解液中荧光素酶活性。

【结果】通过酶切鉴定及测序证明扩增的人PEPCK启动子片段成功插入载体质粒中。

转染后人PEPCK启动子基因荧光素酶活性较空白对照组增加60倍（P＜0．05）；共转染质粒进HepG2细胞或LO2细胞后，HNF4α均可以增强人PEPCK启动子基因荧光素酶活性，在HepG2细胞，荧光素酶活性是对照组的2．69倍（P＜0．05），而在LO2细胞中，荧光素酶活性是对照组的3．64倍（P＜0．05）；PGC-1α可以明显增强HNF4α对人PEPCK启动子的激活作用，在HepG2细胞，荧光素酶活性是PGL3-HPCK-luc＋HNF4α组的2．01倍（P＜0．05），而在LO2细胞中，荧光素酶活性是PGL3-HPCK-luc＋HNF4α组的2．82倍（P＜0．05）。

而与单一转染组相比，共转染pcDNA3．1-PGC-1α与pcDNA3．0-HNF4α显著增加了PEPCK的mRNA和蛋白表达水平（P＜0．05）。

孕激素受体论文

孕激素受体综述孕激素在女性生殖系统中起着重要的作用，这些生理作用主要是通过与孕激素受体结合表达的。

孕激素受体(progesterone receptor，PR)是核受体超家族成员之一。

主要有两种亚型PRA和PRB。

孕激素受体在介导与调节卵巢、子宫和乳腺的功能及生殖活动中起着关键的作用。

一、孕激素受体的结构和功能。

1.PR的结构孕激素受体(PR)，按照氨基酸序列从N一末端到C一末端可分为A、B、C、D、E、F6个区域(图1)。

当有激囊等配体存在时，与E区的激素或配体结合部位(ligand binding domain，LBD)结合，这个区是结构最大功能最复杂的区域；当无配体存在时，D区即铰链区，可结合热休克蛋白(heat shock protein90，HSP90)，从而阻止了各受体之间形成二聚体。

C区是受体与DNA结合的区域(DNA binding domain，DBD)，能与基因调控区的特定DNA序列结合。

A／B区为高度可变区，是受体与抗体结合的部位。

F区则起调节转录激活的作用。

图12.PR的亚型与功能PR的亚型与功能PR主要包括2种亚型PRA和PRB。

在多数情况下，PRB转录活性较强，而PRA则对PRB的转录活性有抑制作用，同时PRA可使孕激素拮抗剂产生抗雌激素样作用，而PRB则无此作用。

PRA和PRB的不同功能是由其结构的差别引起的：特异性反式激活作用决定簇AF1、AF2、AF3和ID可介导孕激素受体的调节，如上图AF1、AF3和ID位于A/B区，AF2位于E区;AF1、AF2和AF3均可单独激活转录，并对启动子和特异细胞起增效作用;AF1和AF2是PRA和PRB所共有的，而AF3是PRB 特有的，位于PRB的上游区;ID是两者共有的，却只在PRA上起作用，能抑制AF1、AF2、ER，但不能抑制AF3的功能，另外ID有利于PRB与辅助因子(SMRT)结合，而PRA不能有效地募集辅助激动子，故PRB活化转录作用强于PRA;PRA不仅活化转录作用弱，而且还可抑制PRB的功能，并可通过非PR结合的磷酸化途径抑制雌激素受体，雄激素受体，糖、盐皮质激素受体介导的转录作用。

【精选】基于重叠延伸PCR法的定点突变技术 doc资料

基于重叠延伸PCR法的定点突变技术E-mail:daican@live △通讯作者:卢光琇, :mutant location Primers Sequences for primers (listed 5'to 3'-189~-193bp (relative to ATGF ggggtaccCTCGCTGTCGCACTCAGGCTRm CAGTCAACCGCCACAAAATT Fm AATTTTGTGGCGGTTGACTG RcggctagcAACTGGGTAGGGACGAGGAG基于重叠延伸PCR 法的定点突变技术*戴灿苗聪秀卢光琇△(中南大学生殖与干细胞工程研究所,人类干细胞工程研究中心湖南长沙410078摘要:目的:建立一种高效而经济的定点突变方法。

方法:采用重叠延伸PCR 定点突变技术,引物设计时引入目的突变,以前两次PCR 产物为模板,进行第三次PCR ,即可获得突变后的目的DNA 片段。

将此片段连入pMD TM 18-T 载体后测序验证突变结果。

结果:DNA 测序表明,待突变位点已由ATTGG 突变为ATTTT 。

结论:成功实现了目的位点的定点突变,重叠延伸PCR 法是一种高效且经济的定点突变方法。

关键词:重叠延伸PCR ;定点突变中图分类号:Q75,Q78,R392文献标识码:A 文章编号:1673-6273(202103-411-02Site-directed Mutagenesis Based on Overlap Extension PCR *DAI Can,MIAO Cong-xiu,LU Guang-xiu △(Institute of Reproductive and Stem Cell Engineering,Central SouthUniversity,National Engineering and Research Center of HumanStem Cell,Changsha,410078,ChinaABSTRACT Objective:To establish a fast,saving method for site-directed mutagenesis.Methods:Overlap extension PCR was used.Briefly,target mutation was introduced into primers,and the two previous PCR products were used as template for the third PCR.The final PCR segment with target mutant was then cloned into pMD?18-T vector for sequencing.Results:DNA sequencing showed that the target site ATTGG had been changed into ATTTT.Conclusion:Site-directed mutagenesis was successfully implemented based on the overlap extension PCR which is a fast and saving method.Key words:Overlap extension PCR;Site-directed mutagenesis Chinese Library Classification:Q75,78,R392Document code:Article ID:1673-6273(202103-411-02前言定点突变(Site-directed mutagenesis,SDM 是指通过聚合酶链式反应(PCR 等方法在目的DNA 片段的特定位点中引入碱基改变,如插入、缺失、点突变等。

PI3K/AKT通路通过Nrf2途径调控PD-L1在非小细胞肺癌中的表达

《中国癌症杂志》2020年第30卷第6期 CHINA ONCOLOGY 2020 Vol.30 No.6419欢迎关注本刊公众号·论　著·通信作者：黄　诚 E-mail: cheng671@PI3K/AKT通路通过Nrf2途径调控PD-L1在非小细胞肺癌中的表达王　静，陈　洁，胡　春，黄　诚福建医科大学附属厦门弘爱医院肿瘤科，福建厦门，361009［摘要］背景与目的：程序性死亡［蛋白］配体-1（programmed death ligand-1，PD-L1）在肿瘤细胞中的表达对肿瘤逃避机体免疫监视具有重要的意义，其表达通常受MAPK 、PI3K-AKT 或STAT3等多条通路的影响，但这些通路具体经哪个关键环节尚不明确。

拟通过PI3K/AKT 信号通路对肺癌细胞A549、H460中PD-L1表达的调控具体机制进行探究。

方法：使用shRNA 技术选择性地沉默A549、H460细胞中的HEB 、HTF 4、Nrf 2及FOXO 3a 等基因，构建缺陷细胞株；将目的基因PD -L 1的3’UTR 区域构建至pGL3-basic 载体中，通过luciferase 双报告基因体系检测PD -L 1转录激活情况，并使用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction ，RTFQ-PCR ）技术验证细胞内PD -L 1基因转录的情况；通过蛋白质印迹法（Western blot ）检测细胞内PD-L1的总表达情况；免疫细胞与肿瘤细胞共培养，检测Nrf 2基因敲除前后，人外周血单个核细胞（peripheral blood mononuclear cells ，PBMC ）对A549、H460细胞株的杀伤效果。

结果：经10 μg/mL 的insulin 刺激后，A549、H460细胞株中蛋白激酶B （AKT ）基因磷酸化水平显著增强，伴随着PD -L 1基因的转录水平和表达水平显著增强（P <0.001），在HEB 、HTF 4及FOXO 3a 基因敲除的细胞株中，情况相似，而在Nrf 2基因敲除的细胞株A549（A549Nrf 2-）和H460（H460Nrf 2-）细胞株中，PD -L 1的转录和表达水平则与阴性对照组一样未见明显变化（P >0.05）；活性氧（reactive oxygen species ，ROS ）诱导剂isoproterenol 能提高A549、H460细胞株中PD -L 1基因的转录和表达水平，而ROS 在被NAC 解除后，PD -L 1的转录和表达水平显著下调，进一步证明Nrf 2对PD -L 1基因的转录调控作用；wortmannin 能逆转insulin 刺激引起的AKT 磷酸化水平增加，促进PD -L 1基因的转录水平和表达水平下降，表明PD -L 1的调控与AKT 激活程度相关；在insulin 和wortmannin 的分别干预下，A549、H460细胞株中的Nrf 2磷酸化水平能随着AKT 磷酸化水平改变而改变，二者相关系数r 分别为0.86和0.93，为强正相关；Nrf 2敲除后，A549、H460细胞株与PBMC 共培养后凋亡数量显著增加。

ELK1结合位点突变的EGR1启动子载体构建和活性测定

1.2.2脂质体法转染人骨肉瘤细胞
对数生长期的293A细胞计数后，按1×106细胞/孔，种于24孔板内，待细胞融合度为60%时，进行质粒转染。以共转染pGL3-EGR1、pRL-TK和pGL3-EGR1mt、pRL-TK为实验组，pGL3、pRL-TK为对照组，按每孔1µg DNA，用1µlLipofectamineTM2000介导转染细胞，48h后，收集细胞。
ELK1为转录因子，为ETS家族中的重要成员，能与DNA直接结合而起到转录激活作用[6]。可通过与c-fos启动子的血清应答元件、血清反应因子结合而形成三元复合物从而介导基因表达。其活性受磷酸化与去磷酸化影响，其表达与MAPK信号通路有关，包括Erk1/2、p3信号途径[7]。
根据软件分析和文献报道，在已获得的EGR1启动子（-669~+157）中含有3个ELK1结合位点，通过将其中一个结合位点（CCCGCCGGAACAAC）突变，检测下游相关分子的变化，为EGR1在癌症治疗方面的功能和机制研究打下了良好的基础。
2结果
2.1 EGR1启动子突变序列的获得
以原有的启动子片段为模板，利用含有突变碱基的引物进行重叠PCR，分别为596bp和305bp的扩增片段；将得到的扩增片段作为模板进行PCR，获得全长为846bp的含有突变碱基的启动子片段，经测序鉴定正确（图1A、1B、1C）。
M:100bpDNA 标准;1:596bp片段;2:305bp片段;3:846bp的含有突变碱基的启动子片段
图3 突变启动子活性检测
3讨论
EGR1最初是作为调控细胞生长和增殖的因子被发现[8]，促进细胞从G0/G1期进入G2/M期。高表达的EGR1能稳定染色体和抑制增殖，也可以促进分化和细胞凋亡；并能直接调控一系列的肿瘤抑制因子、转录因子、生长因子及其受体等的表达，形成下游信号网络决定细胞命运[3,9]。ELK1为核转录激活因子，ELK1基因编码的蛋白结合细胞核，激活Ras-Raf-MAPK信号通路。通过软件分析和文献报道，结合重叠PCR，对EGR1启动子原序列上含有的ELK1结合位点，如果对其结合位点进行突变，通过双荧光素酶实验证明ELK1结合位点突变后基因启动子活性明显下降，则说明ELK1对EGR1的转录具有调控作用。因此，本文对EGR1启动子相关ELK1位点突变的研究，为下一步对EGR1功能研究提供了实验基础。

5、转录调控

分子机制研究套路（五）转录调控课题：转录因子A对B基因的转录调控1．概念介绍：转录水平的调控是真核生物基因表达调控中重要环节。

真核细胞RNA聚合酶自身对启动子并无特殊亲和力，单独不能进行转录，也就是说基因是无活性的。

因此，转录需要众多的转录因子和辅助转录因子形成复杂的转录装置。

在基因转录起始阶段，通用转录因子协助RNA聚合酶与启动子结合，但其作用很弱，不能高效率地启动转录。

只有在反式作用因子（基因特异性转录因子）的协助下,RNA聚合酶II和TFII才能有效地形成转录起始复合物。

反式作用因子（transactingfactor）在转录调节中具有特殊的重要性。

它是能直接或间接地识别或结合在顺式作用元件8~12bp核心序列上,参与调控靶基因转录效率的一组蛋白质。

这类DNA结合蛋白有多种，能特异性识别这类蛋白的序列也有多种，正是不同的DNA结合蛋白与不同的识别序列之间的空间结构上的相互作用，以及蛋白质与蛋白质之间的相互作用构成了复杂的基因转录调控机制的基础。

在真核生物中转录因子的调控是最重要，也是研究得最多的。

蛋白质相互作用在转录因子活性的调控方面具有重要的意义。

细胞内的反式作用因子都是处于有活性和无活性两种状态，这两种状态是可以转换的。

反式作用因子处于无活性状态时，与之相应的基因就不能表达；反式作用因子处于有活性状态、并与相应的顺式作用元件结合时，就可以促进RNA聚合酶和通用转录因子与相应的启动子结合，形成转录起始复合物。

所以，真核基因的表达调控主要是调节反式作用因子的活性，随后反式作用因子调控基因的转录起始。

转录因子被激活后，即可识别并结合上游启动子元件和增强子，对基因转录发挥调控作用。

大部分转录因子在激活以后与顺式作用元件结合，旦也可能有一些转录因子是先结合DNA,被激活后才发挥调节功能。

增强子和上游启动子元件可以结合一些相同的蛋白质，在不同的基因中，存在着同样的顺式作用元件，这表明一些数量有限的基因调控蛋白控制着真核细胞的基因表达。