|
Generation of iPGC via germplasm in vivo. a Scheme for the iPGC induction in vivo using germplasm. b At the 1-cell stage, zebrafish embryos were injected with buc (200 pg), 13GM (50 pg per fraction), or 9GM (50 pg per fraction) mRNA to induce iPGCs. GFP-UTRnanos3 was used to label putative PGCs. The embryos were photographed at 90% epiboly stage. c Schematic diagram for iPGCT. d Time-lapse of iPGC migration to the genital ridges after iPGCT. GFP-UTRnanos3 was used to label iPGCs. e GFP-UTRnanos3 was used to label PGCs and iPGCs, except that immunofluorescence against Piwil1 was used to visualize endogenous PGCs of wild-type embryos at 30% epiboly. f Number of PGCs in wild-type and iPGCT embryos at different stages. Each point represents an independent sample (n ≥ 6). g iPGCs migrated to the genital ridge of the host embryo. h Success rates of iPGCT and conventional PGCT at 1 dpf and 4 dpf. Each point represents an independent experiment (n ≥ 3). i Immunofluorescence detection of Vasa protein in GFP-UTRnanos3-positive iPGCs of iPGCT embryos at 8 dpf. j Probes were designed at the UTR of mRNA (en_probes) to distinguish neonatal mRNA, and single molecule in situ hybridization was used to detect the neonatal germplasm mRNA of ddx4 and tdrd7a in the GFP-UTRnanos3-positive iPGCs and ePGCs at 1 dpf. A representative example of three replicate is shown. All data are presented as mean values ± SEM. Two-tailed Student’s t-test was used to calculate the P values.
|
