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Graphical abstract
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Figure 1. Schematic comparison of the principle of transgenesis using REMI, I-SceI, and NEXTrans methods
Transgenic plasmids are randomly incorporated at multiple loci in the Xenopus laevis genome by the restriction enzyme-mediated integration (REMI)2 and meganuclease (I-SceI)-mediated3 transgenesis methods, whereas the NEXTrans plasmid with CRISPR-Cas9 is integrated into a novel safe harbor site, the tgfbr2l locus.
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Figure 2. Schematic representation of NEXTrans (New and Easy Xenopus Transgenesis) at a novel harbor site
The NEXTrans plasmid carrying the transgene cassette is co-injected into fertilized Xenopus laevis eggs with Cas9 RNP targeting the tgfbr2l.L and tgfbr2l.S loci (exon 4). The plasmid integrates into the target sites in the zygotic genome in a forward and/or reverse direction by NHEJ repair during the early embryonic stage and can be detected by PCR. Primer pairs were used for PCR genotyping to detect the Tg(NEXT-fgk:egfp) integration as follows: primers 1 and 5 for tgfbr2l.L forward insertion, primers 2 and 5 for tgfbr2l.L reverse insertion, primers 3 and 5 for tgfbr2l.S forward insertion, and primers 4 and 5 for tgfbr2l.S reverse direction (see key resources table).
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Figure 3. Representative transgenic X. laevis founders generated by NEXTrans
(A) Representative photograph of eGFP signals in Xla.Tg(NEXT-fgk:egfp) in a promoter/enhancer manner. Strong eGFP signals were detected in the fin and gill.
(B) Mosaic transgenic Xla.Tg(NEXT-fgk:egfp) embryo expressing eGFP only in the fin edge.
(C) Representative photograph of ubiquitous tdTomato signals in Xla.Tg(NEXT-cmv:tdtomato). Strong tdTomato signals were detected in the whole body.
(D) Half-transgenic Xla.Tg(NEXT-cmv:tdtomato) embryo expressing tdTomato signals only on one side of the body.
(E) Representative photograph of tdTomato signals in Xla.Tg(NEXT-cryga:tdtomato) in a promoter/enhancer manner.
(F) Fully transgenic Xla.Tg(NEXT-cryga:tdtomato) with tdTomato signals detected in both eyes completed the metamorphosis normally. Arrows indicate tdTomato signals in the eye; arrowheads indicate the gill; double arrowheads indicate the edge of the tail fin. Scale bars = 1 mm for (A–E) and 5 mm for (F).
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Figure 4. Representative PCR genotyping for targeted integration of NEXTrans plasmid
PCR genotyping was performed using the primer pairs described in Figure 2 and key resources table to detect Tg(NEXT-fgk:egfp) integration in tgfbr2l loci. The genomic DNA was extracted from F1 siblings (#2) generated by in vitro fertilization. Green arrowhead indicates expected PCR products, suggesting Tg(NEXT-fgk:egfp) was integrated into the tgfbr2l.S locus in the forward direction. Samples 1–4, strong eGFP signals were detected in the gill and the edge of the tail fin; sample 5, wild-type (WT) tadpole. HM, high molecular weight marker; LM, low molecular weight marker in the TapeStation electropherogram.
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Figure 5. Germline transmission of transgenic X. laevis generated by NEXTrans
Representative photographs of F1 offspring of (A) Xla.Tg(NEXT-fgk:egfp), (B) Xla.Tg(NEXT-cmv:tdtomato), and (C) Xla.Tg(NEXT-cryga:tdtomato) embryos. Arrowheads indicate the gill; double arrowheads indicate the edge of the tail fin; arrow indicates the eye with tdTomato signal. Scale bars = 1 mm.
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Figure 1. Schematic comparison of the principle of transgenesis using REMI, I-SceI, and NEXTrans methodsTransgenic plasmids are randomly incorporated at multiple loci in the Xenopus laevis genome by the restriction enzyme-mediated integration (REMI)2 and meganuclease (I-SceI)-mediated3 transgenesis methods, whereas the NEXTrans plasmid with CRISPR-Cas9 is integrated into a novel safe harbor site, the tgfbr2l locus.
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Figure 2. Schematic representation of NEXTrans (New and Easy Xenopus Transgenesis) at a novel harbor siteThe NEXTrans plasmid carrying the transgene cassette is co-injected into fertilized Xenopus laevis eggs with Cas9 RNP targeting the tgfbr2l.L and tgfbr2l.S loci (exon 4). The plasmid integrates into the target sites in the zygotic genome in a forward and/or reverse direction by NHEJ repair during the early embryonic stage and can be detected by PCR. Primer pairs were used for PCR genotyping to detect the Tg(NEXT-fgk:egfp) integration as follows: primers 1 and 5 for tgfbr2l.L forward insertion, primers 2 and 5 for tgfbr2l.L reverse insertion, primers 3 and 5 for tgfbr2l.S forward insertion, and primers 4 and 5 for tgfbr2l.S reverse direction (see key resources table).
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Figure 3. Representative transgenic X. laevis founders generated by NEXTrans(A) Representative photograph of eGFP signals in Xla.Tg(NEXT-fgk:egfp) in a promoter/enhancer manner. Strong eGFP signals were detected in the fin and gill.(B) Mosaic transgenic Xla.Tg(NEXT-fgk:egfp) embryo expressing eGFP only in the fin edge.(C) Representative photograph of ubiquitous tdTomato signals in Xla.Tg(NEXT-cmv:tdtomato). Strong tdTomato signals were detected in the whole body.(D) Half-transgenic Xla.Tg(NEXT-cmv:tdtomato) embryo expressing tdTomato signals only on one side of the body.(E) Representative photograph of tdTomato signals in Xla.Tg(NEXT-cryga:tdtomato) in a promoter/enhancer manner.(F) Fully transgenic Xla.Tg(NEXT-cryga:tdtomato) with tdTomato signals detected in both eyes completed the metamorphosis normally. Arrows indicate tdTomato signals in the eye; arrowheads indicate the gill; double arrowheads indicate the edge of the tail fin. Scale bars = 1 mm for (A–E) and 5 mm for (F).
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Figure 4. Representative PCR genotyping for targeted integration of NEXTrans plasmidPCR genotyping was performed using the primer pairs described in Figure 2 and key resources table to detect Tg(NEXT-fgk:egfp) integration in tgfbr2l loci. The genomic DNA was extracted from F1 siblings (#2) generated by in vitro fertilization. Green arrowhead indicates expected PCR products, suggesting Tg(NEXT-fgk:egfp) was integrated into the tgfbr2l.S locus in the forward direction. Samples 1–4, strong eGFP signals were detected in the gill and the edge of the tail fin; sample 5, wild-type (WT) tadpole. HM, high molecular weight marker; LM, low molecular weight marker in the TapeStation electropherogram.
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Figure 5. Germline transmission of transgenic X. laevis generated by NEXTransRepresentative photographs of F1 offspring of (A) Xla.Tg(NEXT-fgk:egfp), (B) Xla.Tg(NEXT-cmv:tdtomato), and (C) Xla.Tg(NEXT-cryga:tdtomato) embryos. Arrowheads indicate the gill; double arrowheads indicate the edge of the tail fin; arrow indicates the eye with tdTomato signal. Scale bars = 1 mm.
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