Amplicap sp6 high yield message maker kit

Myc-nedd8 and GFP were subcloned into Psp64 poly (A) vector (Promega). AmpliCap SP6 High Yield message maker kit (Epicenter) was used for capped mRNA synthesis. Myc-nedd8 and GFP mRNA were synthesized and injected into zebrafish embryos at one-cell stage (400 pg/per embryo). To confirm expression of injected mRNAs, the embryos injected with Myc-nedd8 mRNA for 3 days were harvested and the expression of Myc-nedd8 was confirmed by Western blot using anti-Myc antibody (9E10, Santa Cruz).

The two homologous arms 5’ (738-bp) and 3’ (714-bp) were produced by PCR amplification from the goat genome. The 2,100-bp hLA DNA sequences (from signal peptide to stop codon) were acquired from the human blood genome. The neomycin resistance gene (neo), driven by a phosphoglycerol kinase (PGK) promoter, was designed for positive clone selection and was flanked by two loxP sites which would resulted in the removal of neo sequence by cre-mediated recombination. The hLA-neo cassette was flanked by homologous arms in the PloxpII-hLA-neo vector (Fig 1A). A TALEN1/2 pair targeting exon1 of the BLG gene (GenBank: Z33881) was designed as our previous study[21 ]. Briefly, the binding sites were selected by the “TAL Effector-Nucleotide Targeter”[24 (link)] and assembled by the “unit assembly” method[25 (link)]. The assembled TALEN vectors were linearized by NotI to be used as templates for the in vitro TALEN mRNA transcription with the AmpliCap^™ SP6 High Yield Message Maker Kit (Epicentre).

Customized TALENs against the β-lactoglobulin gene (BLG, GenBank: Z33881) were designed. The candidate target site was selected by the “TAL Effector-Nucleotide Targeter”44 (link). DNA constructs of TALENs were produced by the “unit assembly” method29 (link). Transfection grade plasmids were prepared with the Promega PureYield Plasmid Midiprep System (Promega). The assembled TALEN vectors were linearized by NotI to be used as templates for the in vitro TALEN mRNA transcription with the AmpliCap™ SP6 High Yield Message Maker Kit (Epicentre). The synthesized mRNA was purified with the RNeasy Mini Kit (Qiagen).