图1.?Morpholino 技术原理。（a）Morpholino与RNA结合。（b）Morpholino 通过阻断翻译过程而发挥作用。（c-e）Morpholino阻断RNA的正常剪接。
图2.?Morpholino Knockdown 靶点位置示意图。The zebrafish gene-X was targeted by three specific morpholino antisense strategies to prevent either the translation of the zebrafish gene (ATG-MO) or proper splicing of exon 3 (E3I3-MO).?
图3.?Panels A through H show lateral views of control MO injected zebrafish embryos (Panel A and Panel E) and embryos injected with? gene-X morpholino oligonucleotides (MO) (Panel B through C, Panel F through G), gene-X-e3i3-MO plus nonmutant zebrafish gene-X? (Panel D and Panel H). Coinjection of nonmutant zebrafish gene-X mRNA rescued U-shaped somites (red arrow) and curved body axis? (blue dotted line) in gene-X morphants at 52 hpf. The bar graph in Panel I shows the percentage of embryos with development defects.Panels J Effectiveness of gene-X knockdown was confirmed by RT-PCR and sanger sequencing. hpf, hours post fertilization.?
图4.? Endogenous shha, ptch1, ptch2, sufu, gli1, gli2a, gli2b, and gli3 in wild-type control and geneX morphants assessed by qRT-PCR (n = 100 individual embryos). geneX fine-tunes Hedgehog patterning activity maybe through a novel regulatory feedback loop.?
图5.?GeneX? knock down inhibits the trunk angiogenesis in zebrafish. (A-D) Representative fluorescent images of zebrafish embryos at 32h post-fertilization (hpf). (C-E) Compared with wild-type control, embryos injected with geneX-MO present a lower number of incomplete ISVs and only occasional sprouts (asterisk) of dorsal aorta. The boxed regions are shown at higher magnification in the right panels. DLAV, dorsal longitudinal anastomotic vessels; ISV, intersegmental vessels; DA, dorsal aorta; PCV, posterior cardinal vein.??
图6. Morpholino knock down of geneX induces potent CNS-specific apoptosis. Wild-type control embryos and embryos injected with geneX-MO were stained with acridine orange (AO) at 32hpf. Apoptotic cells are visible as black spots, and less bright homogenous black staining is unspecific background staining. (A-B) Uninjected wild-type control zebrafish exhibited few or no apoptotic cells in CNS (central nervous system). In contrast, significantly increased staining was observed throughout? the CNS in zebrafish injected with geneX-MO (C-D). The blue boxed regions are shown at higher magnification in the right panels. A-D: lateral view, anterior, left.?
图7. GeneX knock down induces potent ototoxicity in zebrafish. Wild-type control embryos and embryos injected with geneX-MO were stained with the mitochondrial potentiometric dye DASPEI at 6-dpf. Hair cells stereotypically located on the lateral line were stained as green dots (white arrow). Uninjected wild-type control zebrafish exhibited normal hair cell number. In contrast, significantly decreased hair cell staining was observed in zebrafish injected with geneX-MO. Fluorescent DASPEI images were inverted for particle analysis. The fluorescence particle signal was quantified using morphometric analysis. dpf, days post fertilization.?
Wang MS, Zhang RW, Su LY, et al. Positive selection rather than relaxation of functional constraint drives the evolution of vision duringchicken domestication. Cell Res. 2016 May;26(5):556-73.?
图8. Nonmutant bmp10 overexpression causes development defects in zebrafish.? (A-C) Gross morphology at 32hpf. Compared with uninjected wild-type control embryos, nonmutant zebrafish bmp10 overexpression causes decreased body length (black dotted line) and curved body axis (blue dotted line) in zebrafish . The bar graph in Panel D through E show the percentage and body length of embryos with development defects. hpf, hours post fertilization.?
图9. Nonmutant bmp10 overexpression inhibits the trunk angiogenesis in zebrafish.? (A-F) Representative bright field and fluorescent images of zebrafish embryos at 32h post-fertilization (hpf). Red arrow indicates haemorrhage in the tail (B). (C-H) Compared with wild-type control, nonmutant? zebrafish bmp10 mRNA (200pg) injection present a lower number of incomplete ISVs and only occasional sprouts (asterisk) of dorsal aorta. The red boxed regions are shown at higher magnification in the right panels. DLAV, dorsal longitudinal anastomotic vessels; ISV, intersegmental vessels; DA, dorsal aorta; PCV, posterior cardinal vein.?
Wang MS, Huo YX, Li Y, et al. Comparative population genomics reveals genetic basis underlying body size of domestic chickens.J Mol Cell Biol. 2016 Dec;8(6):542-552.?
Feng N, Chen H, Fu S, et al. HIF-1α and HIF-2α induced angiogenesis in gastrointestinal vascular malformation and reversed bythalidomide. Sci Rep. 2016 Jun 1;6:27280.??
利用CRISPR/Cas9技术，针对靶基因序列设计sgRNA， 指导Cas9蛋白在特定基因位点引起DNA双链断裂，在非 同源性末端接合修复断裂DNA的过程中，靶基因碱基突变或缺失被引入到斑马鱼基因组中，最终导致靶基因无法正 常转录翻译，达到基因敲除的目的。 目前我们利用CRISPR-Cas9技术，提供AB品系的基因敲除斑马鱼制备，AB系是最为普遍使用的标准纯遗传背景品系之一。同时也可根据委托方的需求，提供TU、Casper 以及其它遗传背景的基因敲除斑马鱼服务。
图10.?tyr Cas9-KO induces pigmentation defects.
图11. Establishment of TG(fabp10a:EGFP) and TG(zlyz:EGFP)? transgenic zebrafish.