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Fig. 4. Targeted integration of egfp into bcan.S locus and expression of eGFP
(A-E) Representative images of bcan.S:egfp embryos. (A, B), bright field images. (A, B), fluorescence images of (A, B). (CE), magnified view of (A, B). Specific eGFP signal was observed in brain (b) and spinal cord (sc) in NF stage 45 embryos (A, B, C-E). Specific eEGFP signal in notochord (nc) was observed in some embryos (A, D) but not in other embryos (B, E). (F) Expression phenotypes of injected normal embryos were classified at around NF stage 43. Br/Sc + NC, specific expression in brain, spinal cord and notochord. Br/Sc, specific expression in brain and spinal cord but not in notochord. Ectopic, ectopic or ubiquitous expression. Rate of classified embryos is indicated as percentage in graph. Total numbers are indicated at bottom. Combined data from four experiments is shown. (G) PCR analysis of genomic DNAs from un-injected wild-type (wt) and tadpoles expressing eGFP in brain, spinal cord and notochord (13) and tadpoles expressing eGFP in brain and spinal cord but not in notochord (4, 5). Left and right gels show PCR fragments including upstream and downstream junctions, respectively. Primers for upstream (bcan.S gFW and egfp RV1) and downstream (vector FW1 and bcan.S gRV) are shown in Supplemental Table 3. Predicted size of the upstream PCR fragment is 1478 bp if the donor is integrated in the forward direction with no indel. Predicted size of the downstream fragment is 1046 bp. The amplified fragments were sequenced and the sequences obtained are shown in Fig. S9.
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