3) The authors use of bulk segregant analysis to identify beneficial mutations and engineering to recreate strains is impressive.
Together, our data provide evidence that expression of human LRRK2 harboring the G2019S mutation in vivo is sufficient to recreate the slowly progressive degeneration of dopaminergic neurons that forms the hallmark pathology of familial and sporadic PD.
We were able to recreate the naturally occurring mutations S81L and G170R in AGT identified in compound heterozygous patient and to isolate and characterize the heterodimer in the purified recombinant form.
To recreate the sets of putative causal mutations, we replaced the five ancestral alleles in the ancestor with the five putative causal mutations in EvoClone2 to make Recreated2 and the eight putative causal mutations in EvoClone9 to make Recreated9.
We were able to recreate the clumpy morphology of EvoClone9 with mutations in MCK1, IRC8, and GIN4; note that EvoClone3, one of the two other clumpy clones with wild type ACE2, also had a mutation in IRC8.
Six putative causal mutations were identified and engineered into a wild-type strain but were insufficient to recreate the oscillator phenotype.
We identified the mutations that led to increased growth on low sucrose and showed that these were necessary and sufficient to recreate the evolved phenotype in two of the evolved clones.
So he decided to recreate it.
