Figure 1.

CRISPR/Cas9-mediated human IFN-β knock-in at the OVA locus in chicken PGCs. (a) Schematic of the knock-in strategy. The top diagram shows the WT chicken OVA locus. The target single-guide RNA (sgRNA) sequence that is part of exon 2 is denoted by the black bar above the nucleotide sequence. The protospacer adjacent-motif sequence is indicated by the red bar. The OVA initiation codon is shown in uppercase letters. The middle diagram shows the donor construct containing the 5′ and 3′ homology regions (HR), the hIFN-β -bovine growth hormone polyadenylation signal construct, and the PGK promoter that drives the puromycin resistance gene (PGK-Puro^r). The bottom diagram shows the KI allele along with PCR primers P1 to P9 that were used for 5′, 3′, and endogenous OVA assays in this study. (b) PCR amplification of the donor cassette knock-in at the OVA locus in the PGC genome. KI PGCs and their parental untransfected cells (UT) were subjected to nested PCR using primers P1–4 (5′ assay) and P5–8 (3′ assay). The middle lane, labeled M, contains DNA molecular mass markers (1-kbp DNA ladder, Nacalai). PCR amplicons of the expected sizes (2.8 kb for the 5′ assay, and 3.2 kb for the 3′ assay) are indicated by the arrows.