摘要
利用CRISPR／Cas9技术，在斑马鱼Daniorerio干扰素γ诱导蛋白30（ifi3D）基因的第一个外显子处选取靶位点，用PCR法构建gRNA，并进行体外转录。将体外转录的sgRNA和Cas9mRNA显微注射到斑马鱼1细胞期的受精卵中，实现对靶基因郑D的沉默。注射后24h检测发现，基因突变率在79％～95％之间，平均突变率为87．2％。为了获得稳定遗传的基因突变纯合系，将F0代与野生型斑马鱼进行配组，获得了3种突变类型的F1代，其突变率为30％。三种突变类型中突变1和突变2为移码突变，突变3删除了6个碱基，并未造成移码。突变1和突变2的F1代杂合个体生长发育未见异常。将杂合F1代自交，理论上将获得突变纯合个体，但仅在受精2h以内的胚胎中检测到了突变纯合个体，受精24h后的胚胎中，只有野生型和杂合型，未见突变纯合个体。推测ifi3D基因的移码突变造成了干扰素γ诱导蛋白的缺失，导致胚胎死亡。
The interferon-gamma-inducible protein 30 （ifi30） gene, as central regulators of immunity in vertebrates, was knocked out in zebrafish using CRISPR/Cas9 to investigate regulation of the immunity by interferons. The small guide RNAs were designed using CHOPCHOP soft-ware to target the first exon of ifi30 gene, and synthesized with PCR. After transcribed in vitro, the mixed mRNAs of guide RNAs and Cas9 were micro-injected into fertilized eggs. The restricted enzyme Pvu II was used for screening genetic mutation in 24hpf embryos, and the mutation rate was found to be ranged from 79% to 95%, with mean value of 87.2%. In order to obtain stable genetic homozygous mutant lines, the F0 individuals were crossed with wild type zebrafish, and 3 mutant alleles were identified in F1, in which two alleles （named Mutl and Mut2 ） caused frameshifts in ifi30 gene, while the other allele did not cause the stop-gained mutant with 6 bp deletion. The heterozygous individuals with Muff and Mut2 alleles were normal in growth and development, while the homozygous embryos with Mutl and Mut2 allele in F2 generation did not develop into larva, the homozygous mutant genotypes only found in survival embryos after 24hpf. The findings suggested that the functional deficiency ofifi30 may result from frame-shift caused death of the embryos during development.
出处
《水产学杂志》
CAS
2016年第6期26-30,共5页
Chinese Journal of Fisheries
基金
中国水产科学研究院基本科研业务费(2016HY-ZD0301)
农业部948项目(2016-X15)
