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?intestinalis embryos. Although ZFNs can cleave and induce mutations at specific sites within animal genomes, the possibility of mutations being introduced to non-specific sites cannot be ruled out completely (Gupta et?al. 2011). To address this issue, we determined sequences of the off-target sites in the genome of embryos injected with EGFP-ZFNs. In the genome of C.?intestinalis, we found 15 potential off-target sites of EGFP-ZFNs which contained three mismatched nucleotides compared with the on-target site of EGFP-ZFNs used in the in silico search. Seven out of 15 potential off-target sites were selected for analyses because these sites could be amplified by PCR and did not have a detectable polymorphism (Table?2). Among the seven potential off-target sites, we identified two off-target sites that were mutated with lower frequency than the on-target site in the embryos injected with 750?fg Hesperadin each of EGFP-ZFN mRNAs (Table?3). The other sites did not have a mutation. To confirm this, we further sequenced more than 100 clones in two of the non-mutated sites (OffT11-1 and OffT38 in Table?3), and did not detect any mutation (Table?3). About the two off-target sites, we examined whether the frequency of their mutations is dependent on the amount of EGFP-ZFN mRNAs introduced into embryos. buy AZD2281 We analyzed genomic DNAs from embryos injected with different amounts of EGFP-ZFN mRNAs. As a result, mutations on the two off-target sites were induced in a concentration-dependent manner (Tables?4 and 5), supporting that the mutations were introduced by EGFP-ZFNs. We did not detect mutations in either off-target site when EGFP-ZFNs selleck compound be mutated by ZFNs with lower frequency than on-target site. However, not all of the off-target sites with three-nucleotide mismatches were targeted by the ZFNs. The chance of the off-target mutations occurring can be reduced by adjusting the amount of ZFNs introduced into the embryos. In order to learn the effects of ZFNs on the normal embryogenesis of C.?intestinalis, we counted the number of normally or abnormally developed embryos that were injected with different amounts of EGFP-ZFN mRNAs. In this experiment, we used wild type animals that did not have the EGFP gene in their genomes because we wanted to demonstrate the toxicities of EGFP-ZFNs on embryogenesis without the damage from the double-strand break of the genome. The percentage of normally developed embryos was normalized using the score of uninjected control embryos (Fig.?3). When 750 or 1500?fg each of EGFP-ZFN mRNAs was injected, the ZFNs did not have an adverse effect on embryogenesis. A larger amount of ZFNs led to defects in embryogenesis.