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Figure 1. Schematic of the extraction procedure used to effectively genotype embryos. A, Embryos in 0.05X MMR are loaded individually per well of the extraction chip. B, The chip is placed in the ZEG unit and run at 1.8 V for 7.5 min. C, The extracted material is used directly for PCR genotyping and downstream analyses. D, The outcome of the analysis allows identification of embryos of interest, (E) which are then selectively used for phenotyping and/or allowed to develop into adulthood
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Figure 2. Cellular material extracted using the ZEG. Following ZEG extraction of Xenopus tropicalis embryos, selected samples across a range of DNA concentrations were mixed with Trypan Blue (1:1) and imaged; (A) NF 25 - known DNA concentration 32 ng/μL, (B) NF 25 -33 ng/μL, (C) NF 19 - 63 ng/μL, and (D) 0.05X MMR (blank). Few Trypan Blue stained (dead) cells and fragments as well as occasional Trypan-excluding, presumably live cells, are visible in preparations. Scale bar = 100 μm.
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Figure 3. Amplification of ZEG-extracted DNA from NF 19 Xenopus tropicalis embryos. DNA was extracted from NF 19 embryos devitellined (lane 2-4) and the same stage embryos with vitelline membrane left in situ (M; lane 5-7). A 260 bp region was amplified successfully as shown resolved on a 1% agarose gel
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Figure 4. One hundred and fifty base pairs fragments successfully amplified from Xenopus tropicalis DNA extracted using ZEG. DNA was extracted from 3-4 individual embryos at NF 25 (lane 2-4), NF 37 (lane 5-7), NF 42 (lane 8-11), and NF 45 (lane 12-15). A 150 bp target region was amplified and resolved on a 1% agarose gel. DNA from a sacrificed embryo (NF 25) was extracted by the conventional method and used as a positive control (Ctrl)
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Figure 5. Example images of Xenopus tropicalis tadpole. Control, non-extracted (A) and post-ZEG extraction (B) at NF 46 are shown. Animals were imaged (25X) and show no visible phenotypic differences
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Figure 6. Fragments of different sizes amplified to determine integrity of ZEG-extracted gDNA. DNA was ZEG extracted at NF 25, NF 37, NF 42, and NF 45, amplified with primer pair A (150 bp), B (260 bp), C (550 bp) & D (800 bp) and resolved on a 1% agarose gel. Contrast was adjusted to reveal the low intensity spurious bands
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Figure 7. Fragments of different sizes amplified to determine ZEG's sensitivity. DNA was ZEG extracted at NF 37 and amplified with primer pair A (150 bp; lane 3), B (260 bp; lane 5), C′ (580 bp; lane 7), and D (800 bp; lane 9). The samples were resolved on a 1% agarose gel alongside positive control DNA PCR products (Ctrl)
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Figure 8. DNA amplification can be facilitated by Restorase. DNA was ZEG extracted at NF 37 and amplified with primer pair A (150 bp; lane 3-4), B (260 bp; lane 6-7) and C (550 bp; lane 9-10). DNA was amplified with either GoTaq G2 Polymerase (ZEG) or Restorase DNA Polymerase (R). The samples were resolved on a 1% agarose gel alongside positive control DNA PCR products amplified with GoTaq G2 Polymerase (Ctrl)
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Figure 9. Results of the T7 endonuclease I assay performed to identify mutant animals. Xenopus tropicalis embryos (n = 22) were subjected to CRISPR/Cas9 gene editing. DNA was extracted using ZEG at NF 37 and a 260 bp target region was amplified. The amplicons were tested for induced mutations by T7 endonuclease I assay. Samples 8, 9, 13, 15, 18, and 19 showed cut bands denoting potential mismatches as a result of INDELs
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Figure 10. Characterization of INDEL formation using Sanger sequencing and TIDE analysis. Sanger sequencing trace of control (left) and sample 19 (right) ZEG-extracted DNA. The degradation of the sequence at the expected cut site (black line) results from INDELs caused by the successful CRISPR/Cas-induced dsDNA break (A). The sequence trace data was input into TIDE to identify the INDELs in the sample (B)
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Figure 11. T7 endonuclease I assay performed with Restorase amplified DNA. Xenopus tropicalis embryos were subjected to CRISPR/Cas9 gene editing. DNA was extracted using ZEG at NF 37 and a 260 bp target region was amplified with GoTaq G2 Polymerase and Restorase DNA Polymerase. The amplicons were tested for induced mutations by T7 endonuclease I assay. Restorase improved amplification while the mutant samples 2 and 3 were accurately detected with both GoTaq and Restorase amplified DNA
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Figure 12. Xenopus laevis DNA amplified following ZEG extraction. DNA extracted from devitellined X. laevis embryos at NF 22 (Lane 2-4) and NF 27 (Lane 5-7), was amplified with primer pair XLA (380 bp amplicon) and the PCR product resolved on a 1% agarose gel. The lower band is non-specific
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