Jesuthasan et al., 2002 - Gene transfer into zebrafish by sperm nuclear transplantation. Developmental Biology   242(2):88-95 Full text @ Dev. Biol.

Fig. 1 Sperm nuclei before (A, B) and after (C, D) demembranation. (A, C) Brightfield images, showing sperm heads on a hemacytometer. (B, D) Fluorescence images, showing Hoechst labeling of nuclei. Only a few nuclei are labeled prior to demembranation, whereas all nuclei are labeled after treatment with lysolecithin. Scale bar, 50 μm.

Fig. 2 Injection method. (A) A schematic diagram of the injection set-up. (B) An egg with an injection needle near the micropyle (arrowhead).

Fig. 3 GFP expression in embryos obtained by sperm-mediated transgenesis or plasmid injection. (A) Lateral view of a 5-h-old embryo fertilized with a sperm nucleus preincubated in DNA encoding histone2B-eGFP. All nuclei appear to fluoresce green. (B) An embryo which had been injected with the histone2B-eGFP plasmid at the one-cell stage. Expression is mosaic. (C) Lateral view of an embryo 24 h after fertilization with a sperm nucleus preincubated in pESG. All cells appear to express GFP, with some, e.g., in the eye, appearing brighter than others. (D) An embryo which had been injected at the one-cell stage with pESG, shown here at 24 h postfertilization. Expression of GFP is widespread, but is not present in all cells. For example, muscle fibers in the plasmid-injected embryos do not express GFP, whereas those of the nuclear-transplanted embryo do. (E) A 5-h-old embryo, derived by transplantation with a sperm head preincubated in pESG. Nuclei are labeled with Hoechst (shown in blue). All cells express GFP, although some express the transgene at a higher level than neighboring cells. (F) An embryo generated by transplantation of sperm nuclei incubated in DNA encoding eGFP under the HuC ΔEco promoter. Bright expression is visible in the forebrain (arrow) and hindbrain (arrowhead), in regions previously shown to express HuC mRNA. Expression in the spinal cord is also visible. The signal in the yolk is due to autofluorescence. (G) An offspring of a fish generated by transplantation of sperm nuclei incubated in ΔEco HuC-eGFP. In this dorsal view of the head of a 24-h embryo, GFP expression can be seen in cells in the forebrain, as well as some migrating neural crest cells (arrowhead). All images are projections of multiple focal planes. Scale bar, 100 μm.

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Reprinted from Developmental Biology, 242(2), Jesuthasan, S. and Subburaju, S., Gene transfer into zebrafish by sperm nuclear transplantation, 88-95, Copyright (2002) with permission from Elsevier. Full text @ Dev. Biol.