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. 2023 Dec 4;15(12):mjad077. doi: 10.1093/jmcb/mjad077

Figure 1.

Figure 1

Derivation of heart-related haploid cell lines from zebrafish embryos. (A) Schematic overview of the derivation of zebrafish parthenogenetic haploid embryonic cells. UV-irradiated sperm were used for the insemination of oocytes, and the zygote cells developing to the blastula stage were dissociated for culture. (B) CCK8 assays demonstrating a higher proliferation rate of the cells cultured with a high concentration (5%) of ZEE. (C) The haploid cells cultured in hCM_BMP4 exhibiting a higher proportion of cells at the G0/G1 stage compared to those cultured in hCM. (D) The proportion of cells at haploid G0/G1 stage in haploid cell lines derived from different strains at passage 5. (E) Phenotype of the haploid cell line PG74_32 derived from a single embryo. The control cell line was a haploid cell line with diploidization. Scale bar, 100 μm. (F) Ploidy analysis of the stable long-term cultured haploid cell line PG74_32 at passage 65. A diploid cell line at passage 35 was used as the control. (G) The 25-chromosome karyogram of PG74_32 at passage 70 arranged in decreasing lengths. (H) UMAP plot of heart-related marker genes expressed in the haploid cell line PG74_32 at passage 70. Two distinct cell clusters were identified, and most of the cells were in one cluster. (I) Whole-mount in situ hybridization was performed to determine krt94 expression in 3 dpf embryos. Black triangles indicate positive signals. Scale bar, 250 μm. (J) Phenotype of krt94-P2A-mCherry knock-in F1 at 3 dpf (the heart region is shown). The fluorescence can be observed at the bulbus arteriosus (BA), ventricular epicardium, ventricular endocardium, and atrial endocardium. V, ventricle; A, atrium. Scale bar, 50 μm. (K) Schematic of the Neon transfection system used to conduct gene editing by RNP. (L) The editing efficiency at the eef1a1l1 locus in PG72ziwi was assessed 3 days after transfection by the T7EI cleavage assay. crRNA–tracrRNA, the pairing of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). %TE, the indel efficiency tested by T7EI.