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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
SouthernBiotech/Goat Anti-Human Lambda-Alexa Fluor® 555/2070-32/1.0 mg
vectorlabs/Biotinylated Aleuria Aurantia Lectin (AAL)/B-1395/1 mg
vectorlabs/VECTASTAIN® Elite® ABC-HRP Kit (Peroxidase, Mouse IgG)/PK-6102/1 kit
vectorlabs/VECTASTAIN® Elite® ABC-HRP Kit (Peroxidase, Universal)/PK-6200/1 kit
vectorlabs/Unconjugated Musa Paradisiaca (Banana) Lectin (BanLec)/L-1410/5 mg
GiottoBiotech/Calmodulin N60D/5 mg/G02CLM60cn