为在斑马鱼中获得特异且高效的基因敲除,多个实验室独立人工合成了序列彼此不一的Cas9 c DNA序列,并克隆入不同的体外转录载体。本文选取两种斑马鱼密码子优化的Cas9编码序列(z Cas9_bz和z Cas9_wc),对斑马鱼胚胎中的7个基因(外源egfp及内源chd、hbegfa、th、eef1a1b、tyr、tcf7l1a)分别进行敲除,通过PCR产物测序、克隆测序和表型分析比较了两种Cas9的敲除效率。结果发现,z Cas9_wc在各种情况下都显现出较高的敲除效率,而z Cas9_bz的效率相对较低。
【英文摘要】
Recent years have witnessed the rapid development of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein(CRISPR/Cas9)system. In order to realize gene knockout with high efficiency and specificity in zebrafish, several labs have synthesized distinct Cas9 c DNA sequences which were cloned into different vectors. In this study, we chose two commonly used zebrafish-codon-optimized Cas9 coding sequences(z Cas9_bz, z Cas9_wc) from two different labs, and utilized them to knocko...
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