By manipulating the various components of the basal organoid media in the context of gene editing, tailored selection for the engineered mutations was accomplished.
Genotyping for the HFE C282Y and H63D mutations was accomplished by polymerase chain reaction (PCR) using validated commercial SNP primers and probes (ABI TaqMan SNP Genotyping Assays C_1085595_10 and C_1085600_10, Applied Biosystems, CA, USA) along with detection using allelic discrimination computation (ABI Prism 7900HT Detection Systems, Applied Biosystems, CA, USA).
Somatic mutation profiling was accomplished by using the Sequenom LungCarta v1.0 panel (http://www.sequenom.com) (Sequenom, San Diego, CA).
Comparable cefotaxime resistant variants were selected relatively quickly (only two rounds of mutagenesis and selection) and with no silent or other superfluous mutations, an approach that was accomplished through what we have termed a "minimal mutational pathway".
The site-specific coupling was accomplished by mutation of single amino acids on GGBP to cysteine and subsequent thiol conjugation.
Assignment of the β3 strand lysine (Lys295 in Src) was accomplished by mutation of Lys295 to methionine in the Src SH3SH2KD protein.
Mutation of calpastatin at cleavage sites of caspases was accomplished using a QuickChange site-directed mutagenesis kit (Stratagene, Santa Clara, CA, USA).
This was accomplished by introgressing the fog-2 (q71) mutation from stock JK574 into each genetic background independently via ten generations of backcrossing.
Domestication, giving rise to organisms with novel combinations of phenotypic traits, was accomplished through human selection for desirable genetic mutations from natural populations.
This was accomplished through finger replacements and key amino acid mutations in the N-terminal fingers, C-terminal fingers, and linker peptide, respectively.
