ProductDescription
DNARepairpathwaysinanimalcellscanbedividedintotwomaincategories: HRandNHEJ. HRorhomologousrecombinationisaminorpathwaybutveryimportantinprotectingcellsfromgenotoxicity. Theprocesshastwokeyrequirementsaswell: ahomologoussequence,usuallyavailableafterDNAreplicationwhenthegenomeis4N,andS-phase. AspecificreporterbasedassayforHRcanbeverybeneficialforanti-cancerdrugdiscoveryprojects,learningmoreabouttheprocessofDNAHRrepairandestablishingintersectingpathwaysanddruggablepathwaytargets.Thisisacell-basedreporterkitdesignedtoallowthecustomertoscreenoridentifyagents(drugs,naturalproducts,smallmolecules,synthetics,miRNAs,andgenes)thataffectorimpacttheprocessofHRDNArepair.ThekitusesGFPasaninvivoreadoutfortheHRpathwayandaplasmidexpressionvectorcontainingthemega-endonucleaseI-Sce1. TheHRreporterplasmidhasbeenengineeredintoamouse3T3celllinewhichisidealforcellcycleanalyses. Thisisbecause3T3cellsnaturallysynchronizeinG1arrest.

DNAiscontinuallybeingexposedtogenotoxicagentsleADIngtocelldeathand/orchangesingeneexpression. OfthevariousformsofDNAdamage,themostdangerousareDNAdouble-strandbreaks(DSBs),whichmaycreateseriousproblemsarisingfrominappropriaterecombinationsuchaschromosomaltranslocations. TodealwiththethreatsposedbyDSBs,cellshavedevelopedmultiplemechanismstodetect,signal,andrepairtheregionsinchromatin. Twomainpathways,homologousrecombination(HR)andnon-homologousend-joining(NHEJ),areinvolvedintherepairofDSB. ThesepathwaysarefurthersuBDividedintomorespecificsub-pathwayprocesses.Inprokaryotes,HRhasbeenknowntobeamajorpathwayfortherepairofDSBs,whileineukaryotes,NHEJwasthoughttobepreferred. Morerecently,HRhasalsobeenshowntooperateinmammals. Thesepathwaysarelargelydistinctfromoneanotherandfunctionincomplementaryways. NHEJinvolvestheligationoftwoDNAendswithouthomologyandtendstobeerrorpronewhileHRishighfidelityandessentiallyerrorfree.IntheHRprocess(sometimesreferredtoasgeneconversion),adonorDNAsequencewithhomologytobothsidesoftheDSBsuppliesgeneticinformationtorepairtheDSB.Thehomologoussequenceiscopiedintothebrokenlocus,makingtherepairedlocusanexactcopyofdonorsequence,withoutalteringthedonorsequence(Fig.1).

Acellbased/cellcontextsystemhasbeendesignedtoallowresearcherstoexamineandinterrogatetheHRprocessinlivecells. TheHRKitusesatwinGFPcassettethatconvertsfromGFPnegativetoGFPpositivecellsusinghomologousrecombination(HR).DNArepairviaHR(asgeneconversion)willresultsinceawildtype(homologous)GFPsegmentispresentincloseproximitytotheDNAbreak. TointroduceapreciseDNAcleavage,amega-endonuclease(I-Sce1)introducesaDSbreakintheGFPlocusofCassette1(Fig.1). ItisimportanttonotethatthemousegenomecontainsnoI-Sce1sites;therefore,anI-Sce1siteinCassette1meansthattheDSbreakoccursonlyatthispreciselocationandnotelsewhereinthegenome. TheDSbreakinitiatesHRandusingtheWTsequenceasahomologytemplate(locatedinCassette2)thegeneconvertstoWTandGFPpositivecellsappear(Fig.2).  HRistriggeredbyaDSbreakwhichisachievedbytransfectingcellswithanexpressionplasmidforI-Sce1(Fig.2).

Thistechnologylendsitselftolivecellimaging(bytrackingsingleGFP+cells). LiveimaginggivesessentiallysinglecellresolutiontoHRanalysisinthesecells. Inaddition,thedescendantsofDNArepaircanbetracked,sincethesecellsarealsoGFPpositive.Seethevideobelowforfurtherdetailsonliveimaging.

 

 

 

KitContents

1. HR-3T3Cellsfrozenincyroprotectionmedium. DeliveredonDryIce. Storeat-80^oCfornotmorethan1weekbeforethawing.
2. I-Sce1geneexpressionplasmid,pSCE(storeat-20^oor4^oC). SeelabelforDNAconcentration.
3. Puromycin.
4. AdetailedprotocoldescribinghowtosetandusethiskitforassayingHRmechanismsinthecellhostprovided.

TopoGen美国TopoGEN公司成立于1991年，是一家诊断公司，由一批在拓扑异构酶领域工作和在基础、临床、应用及相关方面研究经验丰富的研究者创建，为以药物开发为基础的拓扑异构酶和机制研发人员提供创新产品和服务。药物筛选试剂盒可以分析拓扑异构酶活性药物，用于癌症化疗和抗菌方面。TopoGEN公司还能提供原核和真核化验拓扑异构酶 (I型，II，IV和旋转酶)检测试剂盒，体外体内诊断均可应用。TopoGEN公司还提供DNA载体、DNA裂解目标、拓扑异构酶抑制剂、拓扑异构酶抗体和相关试剂盒等完整产品线。TopoGEN is a research diagnostics company that provides innovative products and services for researchers working with topoisomerases and engaged in mechanism based drug discovery and development. Drug Screening Kits are available to identify topoisomerase active drugs for use in cancer chemotherapy and antibacterial applications. The company also provides assay kits for assaying both prokaryotic and eukaryotic topoisomerases (type I, II, IV, gyrase), both in vitro and in vivo.产品列表：No.品牌货号名称规格1TopoGenTG2010-1Monoclonal Antibody to Human Topoisomerase II Alpha50 Units2TopoGenTG2000H-1Human Topoisomerase IIα250 Units3TopoGenTG1025DNA Intercalator/Unwinding Kit50 Experiments4TopoGenTG1001-1Human Topoisomerase II Assay Kit100 Assays5TopoGenTG1010-1SDS-K+ Precipitation Kit (in vitro)100 Assays6TopoGenTG2011-1Polyclonal Antibody to Human Topoisomerase II Alpha250 Units7TopoGenTG2011-3Human Topoisomerase II Alpha Molecular Weight Marker20 Units8TopoGenTG1018-1Topoisomerase I Drug Screening Kit100 Assays9TopoGenTG2005H-RC1Human Topoisomerase I (Top1)250 Units10TopoGenTG2030-1Supercoiled pHOT-1 DNA25 ug11TopoGenTG4110CamptothecinLyophilized12TopoGenTG2035-1Relaxed pHOT-1 DNA25 ug13TopoGenTG406010% Sodium Dodecyl Sulfate1 ml14TopoGenTG4039Proteinase K0.5ml15TopoGenTG4010Topoisomerase I Assay Buffer1 ml