Description: -VijaiKrishnanPh.D,LouisianaStateUniversity -HARISHN.RAMANATHANPh.D.,NIDDK,NIH -RadmilaHrdlickova,Ph.D.,UTAustin -DorisBenbrook,Ph.D.,OUHSC Yes,IcertainlydidusethatsampleofPepMuteyougenerouslysentus
anditworkedsowellonHepG2cellsthatIhavesinceorderedafullsizeandamusingitexclusivelyforsiRNA! IcomparedtoRNAiMAXandRochesX-tremeGENEproducts,buttheseproductswerenotaseffectiveasPepMute. Thanksagainforthesample. Itwasfantastic! -MatthewJackson, Ph.D.,USDA
PepMutesiRNATransfectionReagent,formulatedfromsimulationofviruscellpenetratingpeptides(CPPs),isatotalnovelsiRNAdeliverytoolwhichprovidesmorethan95%silencingefficiencyat1nMsiRNAinvarietyofmammaliancells.Withourproprietarypeptidesimulationtechnology(PST),hundredsofviralCPPsweresimulated,synthesizedandscreenedforgenedeliveryefficacyinvarietyofmammaliancells(Figure1).PepMutereagentwasthenidentifiedandvalidatedasanexceptionallyefficientvectorforcondensingandtransfectingshort(under100bp)singleordoublestrandednucleicacidssuchassiRNA,miRNAmimicsandDNAoligoestowidespectrumofmammaliancells.
Figure1.AcartoonshowingPepMutesiRNATransfectionReagentwasdevelopedbyPST
Size:
-PepMuteReagent,1.0mL,sufficientfor~1,333reactionsbasedontransfecting0.5~5.0pmolsiRNAormiRNAmimicsin24-wellplate
-PepMuteTransfectionBuffer(5x),formulatedformaximaltransfectionefficiency,8.0mLat5xconcentratedstocksolutiontomake40mLofworkingsolution
Applications:
-siRNAmiRNAmimicsormRNAtransfection
-DNA/siRNAco-transfection
-DNAoligoestransfection
Storage:
Storeat4°CforPepMuteReagentandRTforPepMuteTransfectionBuffer(5x). Ifstoredproperly,theproductisstablefor12monthsorlonger.
Advantages:
-Excellentsilencingatfinal1.0nMsiRNA
-Over95%genesilencinginawidevarietyofcells
-Onetubereactionwitheasystandardprotocol
-CompatIBLewithserumandantibiotics
-ProtocolsadaptedtoHTS
-Verylowcytotoxicity
-Veryaffordable
SilencingEfficacyComparisonbetweenPepMutesiRNATransfectionReagentandLeADIngProducts
Figure2.ExcellentsilencingofendogenouslyexpressedKIF11(alsoknownasEG5)inHEK293cellswith1.0µlofPepMutereagentand0.5pmolEG5siRNAperwellof24-wellplate.KIF11(alsoknownasEG5)encodesamotorproteinthatbelongstothekinesin-likeproteinfamilyinvolvedinchromosomepositioningandbipolarspindleformationduringcellmitosis.AreductioninKIF11levelscausesmitoticarrest.PepMutereagenteffectivelydeliversEG5siRNA(final1.0nM)toHEK293cells,leadingtomorethan80%of"round-up"phenotypeofHEK293cells48hposttransfectionovernegativecontrol(final1.0nMwithshamEG5siRNA)whileleadingsiRNAtransfectinreagents,LipofectamineRNAiMAX(RNAiMAX,1.0nMEG5siRNA)/INTERFERin(1.0nMEG5siRNA)/Dharmafect(10.0nMEG5siRNA)andjetPRIME(20nMEG5siRNA)giveaverage37%,23%,53%and48%ball-shapedphenotyperespectivelyonHEK293cells.Thephenotypeof"rounded-up"293cellswerevisualized(upperpanel)andquantified(lowerpanel)48hposttransfectionwithaNikonmicroscope.
Figure3.SilencingefficiencycomparisonofPepMuteTransfectionReagent(upperpanel)withDharmafect4(middlepanel)andLipofectamineRNAiMAX(RNAiMAX,lowerpanel)siRNATransfectionReagentsonA549cells.siRNAtargetingrenillaluciferaseatdifferentfinalconcentrationsrangingfrom0.5to20nMwasco-transfectedwithrenillaluciferasegene(0.5µgofpRL-CMVDNAperwell)bytheabovethreetransfectionreagentspermanufacturers"protocolsintoA549cellsgrowingona24-wellplate.Renillaluciferaseactivitywasdetermined36hafterpostco-transfectionwithrenillaluciferasedeterminationsystem(Promega).Theluminescencewasmeasuredfrom5.0µloflysateduring10sintegrationwithaluminometer(BeckmanCoulterLD400).Luciferaseactivitywasexpressedaslightunitsintegratedover10s(RLU)andnormalizedpermgofcellproteinbyusingtheBCAassay.Theerrorsbarsrepresentstandarddeviationderivedfromtriplicateexperiments.Luciferase-silencingefficiencywascalculatedrelativetountreatedcells.WhilePepMuteandDharmafect4reagentsdeliveredsignificantgenesilencingfrom1.0nMofrenillaluciferasesiRNA,LipofectamineRNAiMAXgavegoodknockdownonlyafter20nMwhileenhancedgeneexpressionatlowconcentrationofsiRNA(0.5and1.0nMrespectively)wasobserved.
Figure4.PepMuteTransfectionReagentknockeddownstableGFPexpressioninMCF7cell(upperpanel)andU2OScell(lowerpanel)byreverselytransfecting5.0and1.0nMGFPsiRNArespectively.Greenfluorescenceprotein(GFP)wasstablyexpressedinMCF7andU2OScells.siRNAtargetingGFPgene(rightpanel)andashamsiRNA(leftpanel)wereintroducedintoMCF7andU2OScellswithfinal5.0and1.0nMrespectivelybyreversetransfectionwithPepMuteTransfectionReagent.GFPgenesilencingwasmonitored48hposttransfectionbyaNikonfluorescencemicroscope.QuantitativeanalysisshowedthatGFPsiRNAat5.0and1.0nMdeliveredbyPepMutesiRNATransfectionReagentknockeddown90%and95%stablyexpressedGFPinMCF7andU2OScellsrespectively.
DataSheet&Protocol
-AStandardsiRNATransfectionProtocol
-AStandardsiRNA/DNACo-transfectionProtocol
-AReversesiRNATransfectionProtocolforHTS
Torequestafreetrialsample,pleaseCreateAnAccountwithustoenteryourshippingaddressandemailusatorder@Signagen.com
Testimonials:
IhadtriedyourproductpepMutetransfectionreagentonprimaryretinalneuronsandwassatisfiedwiththetransfectionefficiency.Willorder!
IreallyappreciateyousendingmeasampleofPepMutesiRNAreagent.ItestedDNA/siRNAco-transfectionusing293Tcellsandtheresultswerecompletelysatisfactory.Iwasabletoget95%knockdownofmytargetgeneat1.0nMsiRNAaswellasexpressionofplasmidDNAusingtherecommendedprotocol.Iwillhavetotestandseeifothercelllinesarealsoaseffective.IwilldefinitelythinkaboutmovingtousethisreagentoverDharmafectorOligofectamine.
ItriedPepMutereagentandIlikeit.Ithassohighefficiencyandnocytotoxicity.Iamgoingtouseit.Thankforintroducingittoourlab.
Tried3differentsiRNAswith100%silencingonA2780cell.Thatisamazing!Ialreadyplacedanorder.
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GFP发出绿色荧光的原理是Ca离子进入GFP的beta-barrel结构中引起的特定能级，因此只要这个结构仍然保持着，就可以发出荧光。
由于GFP的beta-barrel结构非常稳定，一些版本的GFP蛋白（如EGFP）甚至能抵抗94C的高温几分钟而不完全变性，因此想在溶液状态下去掉GFP的荧光是很难的，一般需要用光漂白法。
基于其非常稳定的结构，即便细胞被固定了，仍然会有一部分的GFP蛋白保持其构象而发出荧光。此时荧光可能较弱。在荧光显微镜下是有可能看得到的。
脂质体是磷脂分散在水中时形成的脂质双分子层，又称为人工生物膜。
阳离子脂质体表面带正电荷，能与核酸的磷酸根通过静电作用将DNA分子包裹入内，形成DNA一脂复合体，也能被表面带负电荷的细胞膜吸附，再通过膜的融合或细胞的内吞作用，偶尔也通过直接渗透作用，DNA传递进入细胞，形成包涵体或进入溶酶体 其中一小部分DNA能从包涵体内释放，并进入细胞质中，再进一步进入核内转录、表达。
其次要看下你选择单位的规模如何，上海这边的，你可以看下基尔顿生物，原代细胞培养，动物造模，整体课题外包。
但是有在转染前，将细胞进行重新传代的情况，这样做的目的在于保持细胞的活性状态。
“转染前将细胞以1.5-4.5X104 cells/well 的量(具体接细胞数请参考表1）接种在孔板中，于37℃， 5%CO2 的条件下进行培养，18-24小时（ sf9细胞为3-4小时）后转染。”
脂质体介导法
实验原理
上图所示是脂质体介导转染的示意图，它显示了外源质粒进入细胞的一般过程。
外源基因进入细胞主要有四种方法：电击法、磷酸钙法和脂质体介导法和病毒介导法。电击法是在细胞上短时间暂时性的穿孔让外源质粒进入；磷酸钙法和脂质体法是利用不同的载体物质携带质粒通过直接穿膜或者膜融合的方法使得外源基因进入细胞；病毒法是利用包装了外源基因的病毒感染细胞的方法使得其进入细胞。但是由于电击法和磷酸钙法的实验条件控制较严、难度较大；病毒法的前期准备较复杂、而且可能对于细胞有较大影响；所以现在对于很多普通细胞系，一般的瞬时转染方法多采用脂质体法。
利用脂质体转染法最重要的就是防止其毒性，因此脂质体与质粒的比例，细胞密度以及转染的时间长短和培养基中血清的含量都是影响转染效率的重要问题，通过实验摸索的合适转染条件对于效率的提高有巨大的作用。
上图是本次实验采用的脂质体中阳离子组分的结构的示意图。
本次实验采用的脂质体是promega公司的TransFast脂质体试剂，它是一种阳离子脂质体和中性脂质体的混合物，是对于本次实验中采用的293T细胞优化的转染试剂。
