Knock-in Goat BLG Disruption

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).