凯时K66生物斑馬魚技術服務平台擁有受過嚴格訓練的技術與分析人員，可提供高質量的斑馬魚基因編輯服務，並可通過分析基因敲除、敲低、過表達斑馬魚模型的表型改變研究基因、蛋白在體內的功能以及相關信號通路，篩選致病基因、探索基因功能。

我們提供以下服務：

• 基因Knock-down及表型分析（Morpholino knock-down）
  

• 基因Over-expression及表型分析

• 基因敲除斑馬魚定製（Cas9-KO）

• 轉基因斑馬魚定製

聯繫我們了解更多服務詳情，歡迎來電：400-728-0660，或來函 info@fase-expo.com。

基因Knock-down及表型分析（Morpholino knock-down）

Morpholino knock-down技術原理

圖1. Morpholino 技術原理。（a）Morpholino與RNA結合。（b）Morpholino 通過阻斷翻譯過程而發揮作用。（c-e）Morpholino阻斷RNA的正常剪接。

Morpholino Knockdown應用案例

圖2. Morpholino Knockdown 靶點位置示意圖。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). 

Morpholino Knockdown表型分析

圖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. 

Morpholino Knockdown信號通路機制分析

圖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. 

基因Knockdown後抑制斑馬魚血管生成

圖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.  

基因Knockdown後導致中樞神經系統特異性細胞凋亡

圖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. 

基因Knockdown後具有潛在聽毒性

圖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. 

文獻發表

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. 

基因Over-expression及表型分析

Over-expression 應用案例

圖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. 

圖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. 

文獻發表

1. 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. 

2. 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.  

基因敲除斑馬魚定製（Cas9-KO）

利用CRISPR/Cas9技術，針對靶基因序列設計sgRNA， 指導Cas9蛋白在特定基因位點引起DNA雙鏈斷裂，在非 同源性末端接合修復斷裂DNA的過程中，靶基因鹼基突變或缺失被引入到斑馬魚基因組中，最終導致靶基因無法正 常轉錄翻譯，達到基因敲除的目的。 目前我們利用CRISPR-Cas9技術，提供AB品系的基因敲除斑馬魚製備，AB系是最為普遍使用的標準純遺傳背景品系之一。同時也可根據委託方的需求，提供TU、Casper 以及其它遺傳背景的基因敲除斑馬魚服務。

基因敲除斑馬魚應用案例

凯时K66生物自主研發構建tyr基因敲除斑馬魚。tyr（酪氨酸酶基因）是黑色素合成關鍵酶，該基因敲除後胚胎色素形成受到干擾。

圖10. tyr Cas9-KO induces pigmentation defects.

轉基因斑馬魚定製

轉基因斑馬魚應用案例

利用Tol2轉座子系統構建肝臟特異性EGFP綠色熒光蛋白轉基因TG(fabp10a:EGFP)斑馬魚模型，以及綠色熒光蛋白標記巨噬細胞轉基因TG(zlyz:EGFP)斑馬魚模型。

圖11. Establishment of TG(fabp10a:EGFP) and TG(zlyz:EGFP)  transgenic zebrafish.