|
Fig. 3. Targeted integration of egfp into sox2.L locus and expression of eGFP
(A-F) Representative images of sox2.L:egfp embryos. (AC), bright field images. (A'-C', fluorescence images of (AC). (DF), magnified partial images of (A'-C'). Specific eGFP signal was observed in brain (b), spinal cord (sc), olfactory pit (ol), optic vesicles (op) and otic vesicles (ot) in NF stage 32 embryo (A' D). Signal was additionally observed in gill (g), lenses (le), lateral lines (la) in NF stage 41 (B', E) and NF stage 45 (C', F) embryos. (G) Expression phenotypes of injected normal embryos were classified at around NF stage 40. Specific, brain and spinal cord specific expression. Partial, partial expression in brain and/or spinal cord. Ectopic, ectopic and ubiquitous expression. Rates of classified embryos are indicated as percentage at left side of graph. Total numbers are indicated at bottom. Combined data from six experiments is shown. (H) PCR analysis of genomic DNAs from un-injected wild-type embryos and embryos expressing eGFP in the specific manner. Left and right gels show PCR fragments including upstream and downstream junctions, respectively. Primers for upstream (sox2.L gFW and egfp RV1) and downstream (vector FW2 and sox2.L gRV) are shown in Supplemental Table 3. Predicted size of the upstream PCR fragment is 1384 bp if the donor is integrated in the forward direction with no indel. Predicted size of the downstream fragment is 1275 bp. The amplified fragments were sequenced and the sequences obtained are shown in Fig. S7. The large fragment in lane 4 of the downstream PCR could not be sequenced.
|
|
Fig. 5. Germline transmission of the donor DNA integrated in sox2.L locus.
(A, A' Representative photographs of F1 embryos at 3 days from crossing a wild-type female and a sox2.L:egfp male. Asterisks indicate eGFP-positive embryos. (B, B') Representative images of eGFP-positive (upper) and -negative (lower) embryos at NF stage 40. (C, C) Images of eGFP-positive tadpoles at NF stage 45. (A, B, C) bright field images. (A', B', C') fluorescence images of (A, B, C). Bars, 1 mm. (D) PCR analysis of genomic DNAs from wild-type (wt) and eGFP-expressing embryos. Left and right gels show PCR fragments including upstream and downstream junctions, respectively.
|
|
Fig S6. Sequence of sox2.L fragment in plasmid sox2.L787b-egfp.
Gray and yellow highlighted letters indicate upstream and 5'UTR sequences of sox2.L. Underline indicates sgRNA target sequence with PAM sequence (red underline).
|
|
Fig S7. Sequences of the junction regions between the integrated donor DNA and the targeted sox2.L locus.
Partial sequences of 5' UTRs including upstream (upper) and downstream (lower) junctions from the four embryos expressing eGFP in the specific manner are shown with the wild-type sequence. Downstream junction sequence for embryo 1 was not amplified by PCR (see Fig. 3F). Sequence of the downstream fragment from embryo 4 could not be determined (See Fig. 3F). 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 5' UTR sequence. Blue arrows indicate the deletion sites with the deleted sequences in parenthesis.
|
|
