CRISPR/Cas9技术敲除斑马鱼cdc42基因对骨软骨发育的影响  

Effects of cdc42 gene knockout by CRISPR/Cas9 technology on osteochondral development in zebrafish

作　　者：杨云飞 李仪 孙先定 陈世荣[1] YANG Yunfei;LI Yi;SUN Xianding;CHEN Shirong(Center of Orthopedic and Joint Surgery,the Second Affiliated Hospital of Chongqing Medical University,Chongqing,400016,China)

机构地区：[1]重庆医科大学附属第二医院骨科关节外科中心,重庆400016

出　　处：《陆军军医大学学报》2024年第22期2485-2492,共8页Journal of Army Medical University

基　　金：国家自然科学基金青年基金(82002307);国家自然科学基金面上项目(82372397);重庆市自然科学基金面上项目(cstc2021jcyj-msxmX0121);重庆英才计划“包干制项目”(cstc2022ycjh-bgzxm0052)。

摘　　要：目的利用CRISPR/Cas9技术敲除斑马鱼cdc42基因,探讨其对早期骨软骨发育的影响。方法通过多序列比对分析cdc42的种属保守性,设计cdc42基因的gRNA,利用CRISPR/Cas9技术构建cdc42敲除的斑马鱼突变体。应用整胚原位杂交检测cdc42的表达模式,繁殖并利用转基因标记鱼系Tg(col2a1a:GFP)观察软骨发育表型,并通过茜素红染色观察椎体矿化。结果多序列比对分析显示cdc42在人、小鼠和斑马鱼中高度保守,原位杂交结果显示cdc42在斑马鱼头颅及身体中广泛表达,包括下颌软骨区域。成功设计cdc42的gRNA并利用CRISPR/Cas9技术构建了cdc42基因敲除的斑马鱼突变体。cdc42突变体在受精后第3天表现出体长短小(P<0.01)和头颅发育迟缓,头部和眼睛小(P<0.01),以及下颌发育凹陷。Tg(col2a1a:GFP)斑马鱼显示突变体梅克尔软骨和角舌软骨细胞形态异常,软骨细胞排列紊乱,其角舌软骨夹角增大、长度缩短(P<0.01)。纯合突变体在受精后10~13 d死亡。茜素红染色结果提示突变体椎体矿化数量减少以及软骨内骨化面积减少(P<0.01)。结论通过CRISPR/Cas9技术成功敲除了斑马鱼cdc42基因,导致下颌软骨发育迟缓、椎体矿化延迟以及软骨内成骨减少。Objective To knock out cdc42 gene in zebrafish using CRISPR/Cas9 technology,and investigate the effect of cdc42 on early osteochondral development.Methods After the conservation of cdc42 gene sequence of different species was analyzed by multiple sequence alignment analysis,guide RNA of cdc42 gene was designed,and cdc42 knockout zebrafish was constructed by CRISPR/Cas9 technology.The expression pattern of cdc42 was detected by whole-mount in situ hybridization,and the chondrogenesis phenotype was observed by transgenic labeled fish line Tg(col2a1a:GFP),and vertebral mineralization was observed by alizarin red staining.Results Multiple sequence alignment analysis showed that cdc42 was highly conserved in human,mouse and zebrafish,and in situ hybridization results showed that cdc42 was expressed in a variety of tissues in the head and whole body,including mandibular cartilage.With the aid of guide RNA of cdc42,cdc42 knockout zebrafish was successfully constructed by CRISPR/Cas9 technology.The cdc42 mutants exhibited shortened body length(P<0.01)and delayed cranial development at 3 d post fertilization,with small heads and eyes(P<0.01),as well as delayed mandibular development.The Tg(col2a1a:GFP)zebrafish showed that the mutants presented abnormal morphology of Meckel’s cartilage and ceratohyal cartilage cells,with disordered arrangement of chondrocytes and increased angle and decreased length in ceratohyal cartilage(P<0.01).The homozygous mutants died at 10~13 d after fertilization.The results of alizarin red staining suggested delayed vertebral mineralization and reduced endochondral ossification of the mutants.Conclusion CRISPR/Cas9 technology successfully knocks out the cdc42 gene in zebrafish,resulting in delayed development of jaw cartilage,delayed mineralization of the vertebrae,and decreased endochondral ossification.

关 键 词：CDC42 复发性多软骨炎 CRISPR/Cas9 骨软骨发育 

分 类 号：R322.71[医药卫生—人体解剖和组织胚胎学] R394-33[医药卫生—基础医学] R394.1