Figure 1.
Schematic diagrams showing a PCR-based protocol for identifying mutations induced by TALEN and Cas9 in zebrafish.
(A) Primers designed for detecting mutations in target site. (B) Procedure for identifying induced mutation in zebrafish. WT, wild-type. MT, mutant.
Figure 2.
Determination of known mutations with qPCR.
(A) Relative levels of Po or Pf PCR products. The amounts of Po or Pf PCR products of mixed templates were compared to the amount of corresponding PCR products of pure wild-type template. (B) Relative ratios between Po PCR products and Pf PCR products. The ratios between Po PCR products and Pf PCR products of mixed templates were compared to the ratio of wild-type template. WT, wild-type. MT, mutant.
Figure 3.
Identification of mutations induced by ldlr TALEN.
(A) A schematic diagram showing primers for detecting mutations in TALEN target sites of ldlr gene. (B) The relative amplification efficiency of ldlr Pf primers to Po primers. ldlr TALEN mRNAs injection dramatically reduced the amplification efficiency of ldlr Pf primers. 5 embryos from the wild-type group or the ldlr-TALEN-injected group were pooled and analyzed by qPCR, and 3 pools of each group were analyzed in an independent experiment. Similar results were obtained in three independent experiments. (C) Identification of a mutation in a single allele with T-CIA. Upper panel was an agarose electrophoresis result of PCR products amplified with ldlr Po and Pf primers. Lower panel listed the sequences of mutant colonies identified in upper panel.
Figure 4.
Identification of mutations induced by nsd2 Cas9.
(A) A schematic diagram showing primers for detecting mutations in Cas9 target site of nsd2 gene. (B) The relative amplification efficiency of nsd2 Pf primers to Po primers. nsd2 Cas9 mRNA injection dramatically reduced the amplification efficiency of nsd2 Pf primers. 5 embryos from the wild-type or the nsd2-Cas9-injected group were pooled and analyzed by qPCR, and 3 pools of each group were analyzed in an independent experiment. Similar results were obtained in three independent experiments. (C) Identification of a mutation in a single allele with T-CIA. Upper panel was an agarose electrophoresis result of PCR products amplified with nsd2 Po and Pf primers. Lower panel listed the sequences of mutant colonies identified in upper panel.