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Figure 4 | BMC Biotechnology

Figure 4

From: A single blastocyst assay optimized for detecting CRISPR/Cas9 system-induced indel mutations in mice

Figure 4

Detection of CRISPR/Cas9-induced homologous recombination in murine Ramp2 at the single blastocyst level. A: Schema of the R2gRNA and R2HR DNA donor targeting sites in exon 1 of the murine Ramp2 gene. The R2gRNA-coding sequence is shown in blue. The PAM sequence is shown in red. The R2HR DNA donor comprises 1-kb 5′ and 3′ arms corresponding to exon 1 in the Ramp2 gene and an EGFP expression cassette. Arrows indicate the locations of the PCR primers (see Table 3). B: Fluorescence and bright-field photography of blastocysts developed from fertilized eggs after microinjection (Table 2, experiment 3). C: Gel electrophoretic pattern of PCR products corresponding to the junctional region between the 5′ (or 3′) arm and the host genome. Lanes 1–7: PCR products derived from the crude DNA of individual blastocysts. Lane NC: PCR products derived from the crude DNA of a single uninjected control blastocyst. + and –: EGFP-positive and EGFP-negative blastocysts, respectively. Note that PCR products (approximately 1.1 kb) were successfully amplified only in samples from the 4 EGFP-positive blastocysts.

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