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. Author manuscript; available in PMC: 2015 Mar 6.
Published in final edited form as: Cell Stem Cell. 2014 Feb 13;14(3):323–328. doi: 10.1016/j.stem.2014.01.018

Figure 1. MECP2 mutant monkey fetuses generated by TALEN (see also Figure S1 in the supplement).

Figure 1

(A) Schematic of TALEN-targeting sites within monkey MECP2 locus exon 3. The first nucleotides of exon 3 is numbered as 1, the last nucleotides of exon 3 is number 351. TALEN-targeting sequences are labeled as T1, T2, T3 (L, R). (B) Summary of embryo transfer (ET) after TALEN injection in both rhesus and cynomolgous monkeys (#, embryos transferred between 2-cell and blastocyst stage; a, including 1 singleton and 1 twin; b, miscarriage happened on the 30th and 63rd/64th days after ET; c, one female cynomolgus was born alive after 162 days of gestation; d, one male cynomolus fetus was miscarried on the 92nd day of gestation). (C) Images and gender identification of the two aborted Rhesus monkey fetuses. (D) Paternal and maternal DNAs were subjected to Sanger sequencing and T7EN1 cleavage assays. Since the paternal allele is homogenous, T7EN1 is incapable to cut. The maternal alleles are heterogeneous (with one 217C allele and one 217T allele), thus in T7En1 cleavage assay, positive digestion was observed (two arrows point at cleaved products). (E) Positive T7EN1 cleavage results from the miscarried monkey fetuses’ tail, brain, and testis tissues, indicative of presence of mutations at the targeted exon (cleaved products are indicated by the bracket with a star). (F) All detected point mutations were plotted based on its position and mutation rate in the entire 351bp exon. Mutation distribution plots were displayed for tail, brain and testes of the two miscarried fetuses. (G) Detrimental mutations (leading to early truncations or in-dels) detected in brain and tails of the fetuses. (H) Mutation rates calculated from Sanger sequencing of MECP2 exon 3. From total clones sequenced, we counted perfectly matched sequences with reference and sequences with at least one mismatch. When the percentage of perfectly matched sequences were calculated, we simultaneously acquired the mutation rate in each tissues sequenced (see also Table S1 and Figure S2 in the supplement).