Journal: Developmental cell
Article Title: Precise Editing of the Zebrafish Genome Made Simple and Efficient
Figure Lengend Snippet: Reporter knock-in/knock-out alleles at the kcnh6a locus (A) Schematic representation of the genomic structure of the kcnh6a gene, indicating the kcnh6a-int1 TALEN target, and the structures of the donor DNAs (composed in pKH4 vector), highlighting the reporter coding sequences (colored) and translation/transcription termination signal sequences (grey) that are introduced by the donor. Left and right homology arms are bordered by I-Sce I recognition sites in head-to-head orientation (red arrows). Diagnostic primers are depicted: the rP1/kR1 pair specifically amplifies edited alleles, whereas the kF3/kR1 pair amplifies edited and unedited forms of the kcnh6a gene. (B) In vivo I-Sce I-digestion of donor plasmids stimulates genome editing. Individual embryos were injected with kcnh6a (eGFP) or kcnh6a (mCherry) donor DNAs with or without the I-Sce I meganuclease. Edited alleles were detected by PCR with diagnostic primers. (C) Genome editing is enhanced following I-Sce I digestion of donor plasmids, performed either in vivo or in vitro , prior to injection. Zygotes were injected with TALEN RNA and donor plasmid DNA mixed with differing amounts of I-Sce I enzyme on ice until injection (no pre-digestion) or digested in vitro prior to injection (pre-digestion). As a control, in vitro -digested donor plasmid was injected alone. The fraction of edited alleles (detected with the rP1/kR1 primer pair) relative to total kcnh6a alleles (detected with the kF3/kR1 primer pair) present in injected 2 dpf embryos was determined by qPCR. The relative recombination efficiency was determined by normalizing to 1.0 the mean fraction of edited alleles following injection of TALEN RNA and undigested donor plasmid DNA. For each condition, six individual embryos were analyzed (circles) and the mean relative recombination efficiency is indicated (horizontal dash). Unpaired t-test analysis indicated that in vivo or in vitro digestion of donor DNA with 1mU enzyme significantly stimulated the production of edited alleles as compared with untreated donor DNA (p
Article Snippet: For co-injection with I-Sce I, 0.25mU or 1mU I-Sc eI enzyme was mixed with 50pg donor DNA and 100pg total TALEN mRNA in 0.5× I-Sce I buffer (NEB).
Techniques: Knock-In, Knock-Out, Plasmid Preparation, Diagnostic Assay, In Vivo, Injection, Polymerase Chain Reaction, In Vitro, Real-time Polymerase Chain Reaction