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。
Objective To determine the effect of knocking down zebrafish fafl gene by CRISPR/ Cas9 editing technique. Methods gRNA was designed and prepared for the fafl gene of zebrafish, and gRNA was mixed with Cas9 mRNA by mieroinjection 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 fall gRNA and Cas9 mRNA were successfully prepared. The gRNA （gRNA6） located in the exon 6 of fafl could shift the fafl gene into frameshift mutations. The mutation type MU1 was screened out and the somatic eytochrome 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 dpL Conclusion CRISPR/Cas9 knocking out the fall gene produces a new phenotype for zebrafish, with delayed pigment deposition and nodule-like change in tail muscle section.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2017年第17期1709-1714,共6页
Journal of Third Military Medical University
基金
国家自然科学基金面上项目(31171388)
重庆市杰出青年科学基金(CSTC2012jjjq10001)~~
