Extended Data Fig. 2: Development of mouse embryos electroporated with various concentrations of mRNA and transfection efficiency following electroporation of mouse and human embryos.
From: Live imaging of late-stage preimplantation human embryos reveals de novo mitotic errors
a, Mouse embryos were electroporated at 4-cell stage with an in vitro transcribed EGFP mRNA containing a nuclear localisation signal (NLS) at the concentrations shown. Fluorescent and phase-contrast images were taken 24 hrs after electroporation. The number of embryos developing to the blastocyst stage is summarised. n = 10 embryos electroporated per concentration. b, Mouse embryos were electroporated at 4-cell stage with an in vitro transcribed H2B-mCherry mRNA at the concentrations shown. Fluorescent and phase-contrast images were taken 24 hrs after electroporation. The number of embryos developing to the blastocyst stage is summarised. n = 10 embryos electroporated per concentration. c, Schematic of the experimental setup of mouse embryos electroporated with 500 ng/µl H2B-mCherry mRNA followed by 48 hrs culture in Global media to the blastocyst stage. d,e Control mouse embryos cultured without electroporation (n = 33), a mock electroporation group (n = 23), and embryos electroporated with 500 ng/µl H2B mCherry mRNA (n = 32) were immunofluorescently analysed for the expression of (d) Nanog (epiblast molecular marker), (e) Cdx2 (trophectoderm molecular marker) and DAPI nuclear staining. f, Quantification of total cell number; g, Nanog-positive cells; or h, Cdx2-positive cells. No difference in the number of Cdx2-positive and Nanog-positive cells was observed. P > 0.05 NS, not significant, (two-tailed t-test). Scale bars, 30 μm. Error bars represent the mean ± s.d. Data from three independent experiments. Electroporation efficiency in mouse and human embryos. Embryos electroporated with H2B-mCherry were stained with DAPI (nuclear dye) at endpoint of time-lapses imaging. Individual cells were counted and categorized as DAPI-positive and mCherry-positive or -negative. i, j Representative images of mouse and human embryos, respectively, showing nuclear staining (DAPI) and reporter expression (H2B-mCherry). k, l Percentage quantification of electroporation efficiency, showing the proportion of mCherry-positive cells relative to the total DAPI-positive population. Mouse (n = 10) and human embryos (n = 3), Scale bar, 30 μm. Error bar indicates the mean ± s.d.
