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Fig. 2. Targeted integration of egfp into myod1.S locus and expression of eGFP.
(A-C) Representative images of myod1.S:egfp embryo and tadpole. (AC), bright field images. (A'-C'), fluorescence images of (AC). Clear eGFP signals were found in somitic muscles (A, B) and cranial muscles (C). (A, A', B, B'), lateral views. (C, C'), ventral view of head region. h, heart. Bars in A and B, 1 mm. Bar in C, 200 m. (D) Expression phenotypes of injected normal embryos were classified at around NF stage 40. Specific, somite-specific expression in both sides or either side. Partial, partial expression in somites. Ectopic, ectopic or ubiquitous expression. Rates of classified embryos are indicated as percentage in or on left side of graph. Total number is shown below the graph. Combined data from nine experiments is shown. (E) PCR analysis of genomic DNAs from un-injected wild-type and embryos expressing the muscle-specific eGFP. Left and right gels show PCR fragments including upstream and downstream junctions, respectively. Primers for upstream (myod1.S gFW and egfp RV1) and downstream (vector FW2 and myod1.S gRV) are shown in Supplemental Table 3. Predicted size of the upstream PCR fragment is 1308 bp if the donor is integrated in the forward direction with no indel. Predicted size of the downstream fragment is 1601 bp. The amplified fragments were sequenced and the sequences obtained are shown in Fig. S5.
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Fig S4. Sequence of myod1.S fragment in plasmid myod1.S844b-egfp.
Gray and yellow highlighted letters indicate upstream and 5'UTR sequences of myod1.S. Underline indicates sgRNA target sequence with PAM sequence (red underline).
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Fig S5. Sequences of the junction regions between the integrated donor DNA and the targeted myod1.S locus.
Partial sequences of 5 UTRs including upstream (upper) and downstream (lower) junctions from the four embryos expressing eGFP in the muscle-specific manner are shown with the wild-type sequence. Downstream junction sequences for embryo 1 and 3 were not amplified by PCR (see Fig. 2F). Underline indicates the sgRNA target sequence with PAM sequence (red underline). Inserted nucleotides are indicated in red. Images show examples of sequence chromatograms for the junction regions. Indicated sequences are complementary to the 5UTR sequence. Blue arrows indicate the deletion sites with the deleted sequences in parenthesis.
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Fig S10. Comparison of the targeted integration in the myod1.S locus between myod1.S844b-egfp and myod1.S146b-egfp.
Donor DNA with a truncated myod1.S fragment including only the 146b 5'UTR was constructed using a myod1.S146b FW primer (Supplemental table 1) and used for the targeted integration. (A, B) Representative bright field and fluorescence images of myod1.S844b-egfp-injected embryo (A, A') and myod1.S146b-egfp-injected embryo (B, B). Bars, 1 mm. (C) The eGFP expression ratio was compared between myod1.S844b-egfp and myod1.S146b-egfp injections. Results of two independent experiments are shown.
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