Elaswad Ahmed, Khalil Karim, Cline David, Page-McCaw Patrick, Chen Wenbiao, Michel Maximilian, Cone Roger, Dunham Rex
School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University; Department of Animal Wealth Development, Faculty of Veterinary Medicine, Suez Canal University;
School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University; Anatomy and Embryology Department, Faculty of Veterinary Medicine, Cairo University.
J Vis Exp. 2018 Jan 20(131):56275. doi: 10.3791/56275.
The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene function. Gene knockout has been used to study these gene functions in vivo. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a powerful tool used to edit genomic DNA sequences to alter gene function. While the traditional approach has been to introduce CRISPR/Cas9 mRNA into the single cell embryos through microinjection, this can be a slow and inefficient process in catfish. Here, a detailed protocol for microinjection of channel catfish embryos with CRISPR/Cas9 protein is described. Briefly, eggs and sperm were collected and then artificial fertilization performed. Fertilized eggs were transferred to a Petri dish containing Holtfreter's solution. Injection volume was calibrated and then guide RNAs/Cas9 targeting the toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) gene and rhamnose binding lectin (RBL) gene were microinjected into the yolk of one-cell embryos. The gene knockout was successful as indels were confirmed by DNA sequencing. The predicted protein sequence alterations due to these mutations included frameshift and truncated protein due to premature stop codons.
斑点叉尾鮰（Ictalurus punctatus）的全基因组已被测序，这为研究斑点叉尾鮰基因功能带来了更多机会。基因敲除已被用于在体内研究这些基因功能。成簇规律间隔短回文重复序列/CRISPR相关蛋白9（CRISPR/Cas9）系统是一种用于编辑基因组DNA序列以改变基因功能的强大工具。虽然传统方法是通过显微注射将CRISPR/Cas9 mRNA导入单细胞胚胎，但在鲶鱼中这可能是一个缓慢且低效的过程。在此，描述了用CRISPR/Cas9蛋白显微注射斑点叉尾鮰胚胎的详细方案。简要地说，收集卵子和精子，然后进行人工授精。将受精卵转移到含有霍尔特弗雷特氏溶液的培养皿中。校准注射体积，然后将靶向含Toll/白细胞介素1受体结构域衔接分子（TICAM 1）基因和鼠李糖结合凝集素（RBL）基因的引导RNA/Cas9显微注射到单细胞胚胎的卵黄中。通过DNA测序确认插入缺失，基因敲除成功。这些突变导致的预测蛋白质序列改变包括由于过早出现终止密码子而导致的移码和截短蛋白。
