Twinkle Mutants K319E sample shows decreased mtDNA copy numbers after 24 hours dox treatment Observed in combing experiments and Antoine’s qPCR data Dosed.

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Presentation transcript:

Twinkle Mutants K319E sample shows decreased mtDNA copy numbers after 24 hours dox treatment Observed in combing experiments and Antoine’s qPCR data Dosed with: Dox for 14 hours CldU for 15 min IdU for 60 min media for 90 min

MH-6 K319E-3 In process of quantitating the number of labeled molecules compared to the yoyo images

MH6 - no dox

MH-6 14 hour dox

K319E-3 No Dox

K319E-3 14 Hour Dox

Twinkle Statistics MH6 No dox MH6 14 hr dox K319E3 No dox Group 1 – 35 (61%) Group 2 – 22 (39%) MH6 14 hr dox Group 1 – 92 (75%) Group 2 – 30 (25%) K319E3 No dox Group 1 – 51 (61%) Group 2 – 32 (39%) K319E3 14 hr dox Group 1 – 36 (71%) Group 2 – 15 (29%)

Overexpressing TWINKLE Altered the Replication Profile Group 1 CldU IdU Group 2 We have observed using our single molecule assay, that the overexpression of this protein induces its own phenotype in both the WT and mutant cell lines, not previously observed in less sensitive methods of interrogation. Therefore, we need a cleaner genetic system to investigate our hypothesis, so we are using CRISPRs to create inducible knockouts of endogenous Twinkle IdU CldU IdU