Various knock-in patterns generated by a two-cut HIT-trap donor. (A) PCR genotyping of the puromycin-resistant clones by targeted trapping of Nes and Olig2 in mESCs. The lanes in red rectangles indicate desired 2C knock-in patterns. (B) Schematic showing the desired and alternative knock-in patterns by a two-cut donor (HIT-trap-pA2C). SV40-Puro-IRES-SD (poly(A)-trap cassette), SV40 promoter driving expression of a puromycin-resistance gene followed by sequences of an internal ribosome entry site (IRES) and a splice donor (SD). (C) PCR genotyping verified the predicted alternative knock-in patterns (1C-1 and 1C-2) at the Nes and Olig2 loci in mESCs. The lanes in red rectangles indicate the defined alternative knock-in pattern. (5/3J) 5′/3′ junction. (D) Distribution of different knock-in patterns by HIT-trap-PA2C. (n) Number of clones checked by PCR; (ND) not determined.
