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Fig. 4 | BMC Genomics

Fig. 4

From: One-step generation of a targeted knock-in calf using the CRISPR-Cas9 system in bovine zygotes

Fig. 4

One BTA 17 homolog identified as the map location for the SRY insert in the CRISPR-targeted knock-in bull calf by dual color fluorescence in situ hybridization (FISH). FISH with the donor plasmid (SRY-GFP Anti-Digoxigenin-Fluorescein) as the probe identified one acrocentric chromosome with a positive signal at the q-arm terminal region confirming a single insertion site into the knock-in calf genome. The acrocentric was identified as BTA 17 utilizing a chromosome-specific centromere-proximal probe labelled with Red dUTPs (CHORI BAC 371i17, see Methods). The q-arm terminal location of the SRY green signal found opposite to the centromere proximal BAC red signal compliments the expected insertion location at the safe-harbor as per the sequencing results. Male and female controls (no insertion) were also examined using the same SRY probe with no signal(s) observed (data not shown). a Diploid mitotic metaphase chromosome spread from a fibroblast culture derived from the SRY-GFP knock-in bull shows a normal karyotype, 2n = 60 with a single SRY-GFP positive signal (green arrow) on one of the two BTA17 chromosomes (red arrows) b enlarged SRY-GFP knock-in BTA 17 chromosome (red and green signals) along with the other BTA 17 chromosome (red signal only) from cell depicted in (a) and c, d, e enlarged BTA 17 chromosomes from other cells illustrate the reproducibility of the FISH results. Chromosomes shown in b-e were all enlarged to the same degree

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