Alles N et al. (2023), Automated multi-sample DNA extraction for genot...

XB-ART-59400

Dev Dyn 2023 Mar 01;2523:429-438. doi: 10.1002/dvdy.544.

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Automated multi-sample DNA extraction for genotyping live Xenopus embryos.

Alles N , Guille M , Górecki DC .

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BACKGROUND: Xenopus frogs are used extensively for modeling genetic diseases owing to characteristics such as the abundance of eggs combined with their large size, allowing easy manipulation, and rapid external embryo development enabling the examination of cellular and phenotypic alterations throughout embryogenesis. However, genotyping of mutant animals is currently done either as part of a large group, requiring many embryos, or late in development with welfare effects. Therefore, we adapted the Zebrafish Embryonic Genotyper for rapid genomic DNA extraction from Xenopus tropicalis and Xenopus laevis at early stages. RESULTS: Sufficient and good quality DNA was extracted as early as the Nieuwkoop and Faber stage 19 and, importantly, no detrimental effects of the extraction process on the subsequent tadpole development, behavior, or morphology were observed. Amplicons of up to 800 bp were successfully amplified and used for further analyses such as gel electrophoresis, T7 endonuclease I assay and Sanger sequencing. CONCLUSION: This method allows rapid genotyping during the early stages of Xenopus development, which enables safe identification of mutants. These can be analyzed at early developmental stages or selected for raising without the need for invasive genotyping later, with resource savings and ethical gains in line with the 3Rs principles.

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Species referenced: Xenopus tropicalis Xenopus laevis
Genes referenced: dmd mrc1
GO keywords: CRISPR-cas system

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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
	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 32âng/Î¼L, (B) NF 25 -33âng/Î¼L, (C) NF 19 - 63âng/Î¼L, 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 = 100âÎ¼m.
	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 260âbp region was amplified successfully as shown resolved on a 1% agarose gel
	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 150âbp 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)
	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
	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 (150âbp), B (260âbp), C (550âbp) & D (800âbp) and resolved on a 1% agarose gel. Contrast was adjusted to reveal the low intensity spurious bands
	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 (150âbp; lane 3), B (260âbp; lane 5), Câ² (580âbp; lane 7), and D (800âbp; lane 9). The samples were resolved on a 1% agarose gel alongside positive control DNA PCR products (Ctrl)
	Figure 8. DNA amplification can be facilitated by Restorase. DNA was ZEG extracted at NF 37 and amplified with primer pair A (150âbp; lane 3-4), B (260âbp; lane 6-7) and C (550âbp; 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)
	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 260âbp 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
	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)
	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 260âbp 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
	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 (380âbp amplicon) and the PCR product resolved on a 1% agarose gel. The lower band is non-specific

References [+] :

Blitz, Biallelic genome modification in F(0) Xenopus tropicalis embryos using the CRISPR/Cas system. 2013, Pubmed, Xenbase