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.

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