[1]刘菁,梁森,袁志,等.CRISPR/Cas9技术敲除faf1基因对斑马鱼软骨及肌节发育的影响[J].第三军医大学学报,2017,39(17):1709-1715.
 LIU Jing,LIANG Sen,YUAN Zhi,et al.Effects of faf1 gene knockout by CRISPR/Cas9 on zebrafish cartilage and sarcomere development[J].J Third Mil Med Univ,2017,39(17):1709-1715.
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CRISPR/Cas9技术敲除faf1基因对斑马鱼软骨及肌节发育的影响(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
39卷
期数:
2017年第17期
页码:
1709-1715
栏目:
基础医学
出版日期:
2017-09-15

文章信息/Info

Title:
Effects of faf1 gene knockout by CRISPR/Cas9 on zebrafish cartilage and sarcomere development
作者:
刘菁梁森袁志黄慧哲
重庆医科大学基础医学院:生物化学与分子生物学教研室,发育生物学研究室
Author(s):
LIU Jing LIANG Sen YUAN Zhi HUANG Huizhe

Department of Biochemistry and Molecular Biology, Department of Developmental Biology, College of Basic Medical Sciences, Chongqing Medical University, Chongqing, 400016, China

关键词:
faf1斑马鱼CRISPR/Cas9软骨发育色素沉着延迟肌节异常
Keywords:
faf1 zebrafish CRISPR/Cas9 cartilage development delayed pigmentation sarcoidosis
分类号:
Q75; Q954.48; Q959.468
文献标志码:
A
摘要:

目的     利用CRISPR/Cas9技术敲除斑马鱼faf1基因,并研究faf1基因对斑马鱼发育的影响。方法     针对斑马鱼faf1基因设计并制备gRNA,通过显微注射技术将gRNA与Cas9 mRNA混合注入斑马鱼单细胞胚胎中,通过酶切和基因测序技术筛选出发生突变的F0代斑马鱼,将其与野生型斑马鱼外交得到F1代杂合体斑马鱼,检测其可遗传突变类型,并用显微镜观察、记录每一代斑马鱼表型。结果     成功制备了faf1 gRNA和Cas9 mRNA。位于faf1 6号外显子的gRNA(gRNA6)能使faf1基因发生移码突变。筛选出可遗传突变类型(mutant 1,MU1)并观察到该杂合突变型斑马鱼有体细胞色素沉积延迟,受精后第4天开始尾部肌节出现“结节样”表型以及头颅缩小、舌骨小角角度增大等颅面软骨畸形的变化,并于受精后8~9 d死亡。结论     利用CRISPR/Cas9敲除该基因产生了新的表型,即色素沉积延迟及尾部肌节部位出现“结节样”变化。

Abstract:

Objective     To determine the effect of knocking down zebrafish faf1 gene by CRISPR/Cas9 editing technique. Methods     gRNA was designed and prepared for the faf1 gene of zebrafish, and gRNA was mixed with Cas9 mRNA by microinjection into zebrafish single cell embryos. The mutant F0 generation zebrafish was screened out by enzyme digestion and gene sequencing. The mutant F0 was genetically outcrossed with the wild-type zebrafish to get the F1 heterozygous zebrafish, and the genotype of zebrafish was detected by microscopic observation. Results     The faf1 gRNA and Cas9 mRNA were successfully prepared. The gRNA (gRNA6) located in the exon 6 of faf1 could shift the faf1 gene into frameshift mutations. The mutation type MU1 was screened out and the somatic cytochrome deposition delay was observed in this heterozygous zebrafish. At 4 d post fertilization (dpf), there were sarcomeric dysplasia and head shrinkage, increased hyoid angle and other craniofacial cartilage deformities. And the zebrafish died at 8~9 dpf. Conclusion     CRISPR/Cas9 knocking out the faf1 gene produces a new phenotype for zebrafish, with delayed pigment deposition and nodule-like change in tail muscle section.

参考文献/References:

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更新日期/Last Update: 2017-09-04