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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
matriks/Shikari® (S-ATP) Anti-Pembrolizumab ELISA w/confirmation/PEM-QNS-KEY
intactgenomics/Methionine Auxotrophic LBA4404 | ig® | Intact Genomics/15x50μl/1076-15
goprolytix/Proteolytic Activation of Prothrombin/1 mg, 200 µg/BCT-DFP
novateinbio/Dynorphin A ( 13-17 ) Porcine Peptide/5mg/PE-54024_5mg
matriks/Shikari® (Q-ETA) Etanercept ELISA/ETA-FD-ENB
intactgenomics/Agrobacterium ElectroComponent Combo Pack | Intact Genomics/12x50µl/1290-24
Jackson/Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L)/2.0 ml/115-035-003
Jackson/Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L)/2.0 ml/111-035-003
Qubit™ dsDNA BR Assay Kit Q32853现货
Invitrogen Q32855 Qubit™ RNA HS Assay Kit现货促销
LUCK-1G,D-Luciferin, Potassium Salt 钾盐现货
GoldBio/D-Luciferin, Sodium Salt (Proven and Published™)/LUCNA-1G/1 g