Wang J et al. (2022), Whole-genome sequencing identifies I-SceI-media...

XB-ART-59029

G3 (Bethesda) 2022 May 06;125:. doi: 10.1093/g3journal/jkac037.

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Whole-genome sequencing identifies I-SceI-mediated transgene integration sites in Xenopus tropicalis snai2:eGFP line.

Wang J , Lu C , Wei S .

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Transgenesis with the meganuclease I-SceI is a safe and efficient method, but the underlying mechanisms remain unclear due to the lack of information on transgene localization. Using I-SceI, we previously developed a transgenic Xenopus tropicalis line expressing enhanced green fluorescent protein driven by the neural crest-specific snai2 promoter/enhancer, which is a powerful tool for studying neural crest development and craniofacial morphogenesis. Here, we carried out whole-genome shotgun sequencing for the snai2:eGFP embryos to identify the transgene integration sites. With a 19x sequencing coverage, we estimated that 6 copies of the transgene were inserted into the Xenopus tropicalis genome in the hemizygous transgenic embryos. Two transgene integration loci adjacent to each other were identified in a noncoding region on chromosome 1, possibly as a result of duplication after a single transgene insertion. Interestingly, genomic DNA at the boundaries of the transgene integration loci contains short sequences homologous to the I-SceI recognition site, suggesting that the integration was not random but probably mediated by sequence homology. To our knowledge, our work represents the first genome-wide sequencing study on a transgenic organism generated with I-SceI, which is useful for evaluating the potential genetic effects of I-SceI-mediated transgenesis and further understanding the mechanisms underlying this transgenic method.

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Species referenced: Xenopus tropicalis
Genes referenced: snai2

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	Fig.1. Workflow for detection of integration sites. Workflow detailing each major step in the pipeline of detection of transgene integration sites in the X. tropicalis genome.
	Fig.2. Sequencing depth of WGS reads mapped to the snai2 gene and transgene sequences. a) Sequencing depth of the snai2 gene and surrounding region on chromosome 1. The snai2 promoter, which is also part of the transgene construct, is highlighted in pink, and the transcribed region is shown in green. b) Sequencing depth of different regions of the transgene. Reads with low mapping quality, including those with low complexity and those perfectly mapped to other regions of the genome, have been removed. Y-axis represents sequencing coverage.
	Fig.3. Mapping of the 2 transgene integration loci to chromosome 1 of the X. tropicalis genome. a) Localization of the transgene insertions in chromosome 1. The 100-kb surrounding region (middle) includes the nearby genes, and the insertion region is further zoomed in to show the locations of the 2 transgene integration sites and the genomic DNA sequences at the boundaries (bottom). b) A linear map of the transgene construct. The genomic DNA and transgene sequences are connected with red and green dotted arrows, respectively, at the 2 integration sites (I1 and I2). See Supplementary Fig. 1 for WGS reads that align with the junctions of transgene integration sites. Junction sequences were further confirmed by PCR and Sanger sequencing (see Fig. 4).
	Fig.4. Locations of primers and PCR validation of transgene integration sites. a) Design of PCR primers. Primer pairs 1 and 3 covered the left boundaries of the transgene integration sites (I1 and I2), primer pair 2 covered the right boundary of I2, and primer pair 4 covered the region between I1 and I2 along with the flanking transgene sequences. Green bars represent Xenopus genomic DNA, gray and orange arrows represent the snai2 promoter and eGFP coding sequence from the transgene, respectively, and white blocks represent the vector sequences from the transgene. Segments are separated from each other for better visualization. b) PCR amplifications of junction sequences using the primers shown in (a). c) Sanger sequencing results across the insertion boundaries of I1 (upper) and I2 (lower).
	Fig.5. A model for I-SceI-mediated transgenesis that resulted in the snai2:eGFP X. tropicalis line. Genomic DNA is shown in blue and transgene construct in red. Green and orange boxes highlight the sequence homology between the genomic DNA at the junctions and the I-SceI recognition sequence at the left end of transgene construct and the 3â€²-overhang generated by I-SceI cleavage at the right end, respectively. Purple box highlights the homology between the upstream and downstream genomic sequences at the initial transgene insertion site that possibly led to a subsequent realignment. See Discussion for further explanation.
	Figure S1. WGS reads mapped to the reconstructed Chromosome 1 with the inserted transgene. Soft-clipped reads were aligned to the left (a) and right (b) boundaries of transgene integration site 1 (I1), as well as the left (c) and right (d) boundaries of integration site 2 (I2). X. tropicalis genome, transgene sequence and WGS reads are highlighted with green, purple and gray, respectively.
	Figure S2. Recombination sites of transgene concatemer. Transgene construct was concatenated and inserted into genomic sequence on Chromosome 1. Three intramolecular recombination sites (IR1-3) were supported by WGS reads. The concatemeric joints between two copies are linked by curves of different colors.

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Abrahams, Metaphase FISHing of transgenic mice recommended: FISH and SKY define BAC-mediated balanced translocation. 2003, Pubmed