Generation of a precise Oct4-hrGFP knockin cynomolgus monkey model via CRISPR/Cas9-assisted homologous recombination. (A-C) Detection of Oct4-GFP knockin in abortive fetuses. (A) PCR amplification of a GFP fragment from different tissues of abortive fetuses I, J, K (060366A, B, and C). H, heart; Li, liver; Lu, lung; B, brain; Um, umbilical cord; WT, wild-type control. (B) PCR amplification of a 2 086 bp 5′-arm with part of the GFP and the L-HA (left), or a 2 343 bp 3′-arm with part of the GFP and the R-HA (right). (C) Southern blot analysis of the targeted allele. StuI-digested genomic DNA from small intestine and muscle of fetus J (060366B) was hybridized with different probes (left: Probe 1; middle: Probe 2; right: Probe 3). Expected fragment sizes: 4 719 bp (mutant), 3 392 bp (wild type). hrGFP, mutant fragment detected by hrGFP internal probe (Probe 1); 5′-arm, mutant fragment detected by 5′ external probe (Probe 2); 3′-arm, mutant fragment detected by 3′ external probe (Probe 3). WT, wild-type genomic DNA (negative control); Mu, muscle; SI, small intestine. (D-F) Detection of Oct4-GFP knockin in founder animals. (D) Photos of 403-day-old founder 152008 (left) and 401-day-old founder 152018 (right). (E) Nested PCR amplification of a 1 544 bp 5′-arm with part of the GFP and the L-HA (upper), and a 2 343 bp 3′-arm with part of the GFP and the R-HA (lower) from different tissues of founder animals (152008, 152018). Pl-M, maternal portion of the placenta; Pl-E, embryo portion of the placenta; Um, umbilical cord; Bl, blood; WT, wild-type control. (F) The sequence of the amplified genomic DNA from the boundaries of 5′-arm (upper) and 3′-arm (lower). (G) The sequence of cDNA from the boundaries of Exons 4 and 5 (upper) as well as 3′-UTR and IRES (lower). (H) Western blot analysis of the expression of hrGFP and Oct4. The expression of hrGFP and Oct4 was examined in the ovaries of wild-type control and fetus J (060366B). COS7 cells with or without hrGFP overexpression serve as controls and β-actin was used as a loading control. (I) PCR products of cDNA reversely transcribed from mRNA isolated from the iPSCs of Founder 152008. WT, wild-type somatic cell control. (J) Extracted ion chromatograms of transitions monitored for detection of hrGFP protein in iPSCs generated from founder 152008 and different types of control by mass spectrometry. GNILFGNQLVQIR derived from hrGFP protein along with internal heavy standards were detected. Heavy peptide with BSA, isotope-labeled peptides were spiked into tryptic peptides of bovine serum albumin; wild-type iPS, iPS cells derived from wild-type monkey; GFP iPS, iPS cells with GFP overexpression; 152008 iPS, iPS cells derived from hrGFP knockin monkey 152008; hrGFP COS-7, COS-7 cells with hrGFP overexpression as a positive control. All primers used for PCR amplification were illustrated in Supplementary information, Figure S1 and described in Supplementary information, Table S4.