Description
Basedonourproprietarypolymersynthesistechnology,PolyJetDNAInVitroTransfectionReagentisformulatedasabiodegradablepolymerbasedDNAtransfectionreagentthatensureseffectiveandreproducIBLetransfectiononHEK293,COS-7,NIH-3T3,HeLa,CHOandabroadrangesofhard-to-transfectmammaliancells.PolyJetreagentisabletoimmobilizeDNAmigrationduringelectrophoresisatverylowconcentrationandformtransfectioncomplexwithin5minutesatRT.Aremarkablefeatureofthereagentistherapidandcompletedegradationofpolymeraftertransfectioncomplexendocytosis(Figure1),leADIngtomuchlesscytotoxicity.PolyJetreagent,1.0ml,issufficientfor~667transfectionsin24wellplatesor~333transfectionsin6wellplates,providingaveryaffordablealternativetotheleadingproductsfortransfectingavarietyofcommonlyusedandhard-to-transfectmammaliancells.
Figure1.ACartoonShowingBiodegradationofPolyJetDNATransfectionReagentAfterEndocytosisofTransfectionComplex
Features
-Bio-degradableafterendocytosis
-Exceptionalhightitersofvirusproduction
-EquallygoodforverylongDNAs(>89kb)
-EquallygoodforbothsingleDNAtransfectionandmultiDNAco-transfection
-Highlevelsofrecombinantproteinproduction
-Simple&robusttransfectionprocedure
-Veryaffordable
StorageCondition
Storeat4°C.Ifstoredproperly,theproductisstablefor12monthsorlonger.
BroadTransfectionSpectrumforMammalianCellTypes
CellLines | Efficiency(%GFP) | CellLines | Efficiency(%GFP) |
| McArdle7777 Hep3D SHEP 3T3-442A COS-7 CV-1 D407 DHDPro.b 3LL B16-F10 BAEC BHK-21 CaSki CaCo2 CHO HCS-2/8 HEK-293 HeLa HLMEC H-MVEC Huh-7D12 ATT20 SK-N-SH C2C12 HepG2 | 65-70% | hESCs | 70% |
ExamplesShowingTransfectionEfficiencyofPolyJetDNAInVitroTransfectionReagentonCommonlyUsedCells
TransfectionefficiencycomparisonofPolyJetvs.lipofectaminePlusonChineseHamsterOvary(CHO)cells.HAtaggedbeta-tubulinCDNAwasdeliveredintoCHOcellswithPolyJet(leftpanel)andlipofectaminePlus(rightpanel)respectively.FITCconjugatedantibodyagainstHAtagwasutilizedtopickupHA-beta-tubulin(Green)whileaDM1aantibodywasusedtodetectendogenousalpha-tubulinfollowedbyprobingwithrhodamineconjugatedsecondaryantibody(Red).TheabovepicturewasprovidedbyDr.ShangYinofUniversityofTexasatHoustonMedicalSchoolascourtesy
AcomparisonshowingtransfectionefficiencyofPolyJetreagentvs.aleadingproduct,Lipofectamine2000onHEK293FTcells.HEK-293FTcellsweretransfectedwithGFPvector(pEGFP-N3)byPolyJet(leftpanel)andLipofectamine2000(rightpanel)respectively.ThecellswerevisualizedbyNikonEclipseFluorescencemicroscope24hoursposttransfection
AcomparisonshowingtransfectionefficiencyofPolyJetreagentvs.aleadingproduct,Lipofectamine2000onHepG2cells.HepG2cellsweretransfectedwithGFPvector(pEGFP-N3)byPolyJet(leftpanel)andLipofectamine2000(rightpanel)respectively.ThecellswerevisualizedbyNikonEclipseFluorescencemicroscope24hoursposttransfection
TransfectionefficiencycomparisonofPolyJetvs.FugeneHDonMDCKcells.AplainGFPDNAwastransducedintoMDCKcellswithPolyJet(leftpanel)andFugeneHD(rightpanel)reagentsrespectivelypermanufacturers"protocols.GFPandDAPIstainingwerevisualizedunderfluorescencemicroscopy48hoursposttansfection.TheabovecomparisondataandpictureswerecompletedandprovidedbyDr.GeZhouofNYUMedicalCenterascourtesy
AcomparisonshowingtransfectionefficiencyofPolyJetreagentvs.aleadingproduct,Lipofectamine2000onMDCKcells.MDCKcellsarenotoriouslyhardtotransfect.Withproprietary"ShavedCellTransfection"protocol,PolyJet(leftpanel)givesupto70%GFPpositivecellsvs.Lipofectamine2000(rightpanel)around5%efficiency.MDCKcellsweretransfectedwithGFPvector(pEGFP-N3)byPolyJet(leftpanel)andLipofectamine2000(rightpanel)respectively.ThecellswerevisualizedbyNikonEclipseFluorescencemicroscope36hoursposttransfection
AcomparisonshowingtransfectionefficiencyofPolyJetreagentvs.aleadingproduct,FugeneHDonLNCapcells.LNCapcellsweretransfectedwithGFPvector(pEGFP-N3)byPolyJet(leftpanel)andFugeneHD(rightpanel)respectively.ThecellswerevisualizedbyNikonEclipseFluorescencemicroscope24hoursposttransfection
AimageshowingexceptionaltransfectionefficiencyofPolyJetreagentonHumanembryonicstemcells (hESCs). ThehESCsgrowninE8mediumonGeltrexvs(leftpanel,DICimaging)wastransfectedwithpEF1α-GFP.TheGFPexpression(rightpanel)wasvisualizedbyNikonEclipseFluorescencemicroscope24hoursposttransfection. TheabovepictureswereprovidedbyDr.MarinaPryzhkovaofJohnsHopkinsUniversityascourtesy
Neuro2AcellstransfectedwithpEGFP-C1plasmidusingPolyJetInVitroDNATransfectionReagent.TheNeuro2AcellswerevisualizedbyNikonEclipseFluorescencemicroscopewithDICphaseimaging(left)andFITCimaging(right)24hourspost-transfection
ComparisonofcytotoxicityofPolyJetDNAInVitroTransfectionReagentwithL2Konprimarymurineskinfibroblast.Theprimarymurinefibroblastwasincubatedwiththeindicatedtransfectionreagents/pEGFP-C1(DNA)complexesabovefor4hoursinserum-freeDMEMHighGlucosemediumfollowedbyreplacementofcompleteserum-containingmedium.ThecellswerevisualizedbyNikonEclipseFluorescencemicroscopewithDICphaseimaging24hoursposttransfection
DataSheetandProtocols
-GeneralProtocolforTransfectingMammalianCells
-AShortProtocolforTransfectingMammalianCells
-AdvancedProtocolforTransfectingHard-to-TransfectMammalianCells
-AProtocolforTransfectingSUSPension293andCHOCells
-ProtocolforLentivirusProduction
-ProtocolforrAAVProduction
- TechnicalNote&TransfectionTips
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Testimonials:
Sorryforsooobigdelay,butnowIamtotallyinlovewithPolyJet.IhaveusedPolyJetforawhileonmouseESCswithrelativelygoodefficiency50~70%.
--------MarinaPryzhkova,Ph.D.,JHU
PolyJetTransfectionReagentworkedequallyaswellaslipofectamine2000,withlittleevidenceofcelldeathon293,PC-3and22RV1cells.Iwilldefiantlyconsiderswitchingover.
-----TiffanyWallace,Ph.D.,NCI/NIH
Ionlydidsidebysidewiththetestingsample(PolyJet)andLipo2000withGFPtransfectiononCOS-7cells.Theresultwasverygood.PolyJetwasevenbetterthanL2K.
------FengQiao,Ph.D.,NEI/NIH
ItestedthesampleofPolyJetonmyNIH-3T3mousefibroblaststhisweekend.TheresultsweremuchbetterthanLipofecatmineLTX.I"mattachingapowerpointslidewithmyresults(Ididnotquantifythe%transfectionefficiency,butthepicturesgetthepointacross).Ifoundthattheprotocolfordifficult-to-transfectcelllinesworkedbetterthanthestandardprotocol.
-----StephanieMurphy,Ph.D.,DartmouthCollege
Ihadchancetotryyourproductfinally.Itwasgreatsuccess.IusedHeLacellsandgot10%transfectionefficiency(<0.1%forLipofectamine).Thankyou!IwaswonderingifIalsotryGenJetPlusDNAInVitroTransfectionReagent?Accordingtoyourwebsite,thereagentworksbetterthanregularPolyJet.
-------YumiUetake,Ph.D.,UMASS
ItestedPolyJetanditlooksgreatonMDCK.Weplacedorder.Thankyou!
-------GeZhou,Ph.D.,NYU
Wearehappytoprovidefeedback.PolyJetworkedverywellforusinHepG2cells,wegotapproximately80%efficiencywithpMAXGFPplasmid,byfollowingtheconditionsinyoursuggestedprotocol.WeranacomparisonwithLipofectamine,whichonlyshowedapproximately20-30%transfectionefficiency.Weareplanningexperimentsandwillbeorderingmoresoon.
-------EmilyMcallister,PBRC
ebiomall.com
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DXY721认为：
悬浮细胞和贴壁细胞在转染过程中差别不大，主要差别在于转染后的筛选，当然如果你做的是瞬时转染就不存在筛选的问题了。
其实转染的过程很简单，问题是能不能转的进去的，转染率能有多少，转进去是否可以稳定表达目的蛋白等等。
我们也是用脂质体做悬浮细胞的转染，说明书上都有具体的操作过程，将脂质体和目的基因按比例混合，然后加到细胞悬液里就OK了，说的简单，实际上还是有一些细节要注意的，比如脂质体和目的基因混合的比例，转染的细胞数，细胞的代数，细胞的状态，有的还要求在转染的前一天传代一次，不过不要怕，这些在脂质体说明书上都有明确的说明，按照说明书做就可以了。
jinghuanlv认为：
悬浮细胞和贴壁细胞转染还是有很大不同的。
脂质体转染的原理基于电荷吸引原理，先形成脂质体-DNA复合物，散布在细胞周围，然后通过细胞的内吞作用，将目的基因导入细胞内，而脂质体复合物与贴壁细胞的接触机会比悬浮细胞高出很多倍，所以，脂质体转染时悬浮细胞的转染效率要明显低于贴壁细胞。
我们实验室转染悬浮细胞是用的电穿孔法，目前为止，悬浮细胞转染的最好方法还是电转，我们实验室用的电转仪是Bio-Rad的，使用条件是电压250V，电容975uF，效果不错，不妨一用。
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。
本人研究生
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。
各位版友求助，
我使用Hek293构建转染模型，瞬转5质粒，用lipo2000做转染体系。
转染48h，发现荧光较强的细胞都在爬片的边缘，比例十分少。是因为我添加试剂的手法不对吗。
同时也发现加入转染体系后细胞状态特别差。想问一下用lipo2000时可以用无双抗的10%FBSDMEM吗。我转染前6h现在用的是纯DMEM，不含FBS。
请各位大神帮忙
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。”
