CRISPR/Cas9技术敲除斑马鱼rpl15基因对斑马鱼红系造血发育的影响  

Effects of rpl15 gene knockout by CRISPR/Cas9 on zebrafish erythroid hematopoietic development

作　　者：彭涛 周唯君 刘含 赵砚驰 李化 张鹏 周艳华[1,2] 舒莉萍 PENG Tao;ZHOU Weijun;LIU Han;ZHAO Yanchi;LI Hua;ZHANG Peng;ZHOU Yanhua;SHU Liping(National&Guizhou Joint Engineering Laboratory for Cell Engineering and Biomedicine Technique,Guizhou Provincial Key Laboratory of Regenerative Medicine,Department of Immunology,School of Basic Medicine,Guizhou Medical University,Guiyang,Guizhou Province,550004;Key Laboratory of Adult Stem Cell Translational Research,Chinese Academy of Medical Sciences,Guiyang,Guizhou Province,550004,China)

机构地区：[1]贵州医科大学细胞工程生物医药技术国家地方联合工程实验室,基础医学院免疫学教研室,贵州省再生医学重点实验室,贵阳550004 [2]中国医学科学院成体干细胞转化研究重点实验室,贵阳550004

出　　处：《第三军医大学学报》2021年第8期683-691,共9页Journal of Third Military Medical University

基　　金：国家自然科学基金地区科学基金项目(31860325);贵州省科技计划项目(黔科合基础【2020】IY092)。

摘　　要：目的利用CRISPR/Cas9技术敲除斑马鱼rpl15基因,探讨rpl15基因对斑马鱼红系造血发育的影响。方法针对斑马鱼rpl15基因设计并制备gRNA(guide RNA),将gRNA与Cas9蛋白的混合物显微注射到单细胞期斑马鱼受精卵中,提取72 h胚胎基因组DNA,PCR扩增出目的片段,T7E1限制性酶切法检测基因打靶情况,随后进行测序分析确定编辑效果。通过O-dianisidine染色分析血红蛋白合成情况,中性粒细胞染色分析中性粒细胞生成情况,利用Real-time PCR检测p53、mdm2、hsp70和造血相关转录因子基因的表达变化。结果成功制备了rpl15 gRNA,并筛选得到rpl15突变体。rpl15基因敲除的幼鱼出现体长缩短、尾部弯曲、头部和眼睛较小及心包水肿的表型;造血相关谱系染色结果显示rpl15突变体血红蛋白合成明显减少,但中性粒细胞生成未受影响;rpl15突变体p53和mdm2的mRNA表达均显著升高(P<0.01,P<0.05,),α-globin、gata1和hsp70的mRNA表达均显著下调(P<0.01,P<0.05,P<0.05),除红系外的造血相关转录因子的mRNA表达与对照组相比均无明显差异。结论利用CRISPR/Cas9技术成功编辑了斑马鱼rpl15基因,突变体产生了类似先天性纯红细胞再生障碍性贫血疾病的表型。同时表明p53与hsp70可能参与调控突变体表型的形成。ObjectiveTo determine the effect of knocking out zebrafish rpl15 gene by CRISPR/Cas9 editing technique on zebrafish erythroid hematopoietic development.MethodsGuide RNA was designed and prepared for rpl15 gene of zebrafish and the mixture of gRNA and Cas9 protein was microinjected into zebrafish zygote at single cell stage.Genomic DNA was extracted after 72 h and the target fragment was amplified by PCR.Editing effect was evaluated by T7E1 restriction enzyme digestion of the PCR products followed by DNA sequencing.Hemoglobin synthesis was analyzed by O-dianisidine staining,neutrophil production was analyzed by neutrophil staining,and the expression of p53,mdm2,hsp70 and hematopoiesis related transcription factor genes was detected by real-time PCR.ResultsThe rpl15 gRNA was successfully prepared and the rpl15 mutants were selected.The results showed that zebrafish rpl15 gene knockout juveniles showed a phenotype of shortened body length,curved tail,smaller head and eyes,and pericardial edema.Hematopoietic lineage staining showed that hemoglobin synthesis in rpl15 mutant was significantly decreased,but neutrophil production was not affected.The mRNA expression levels of rpl15 mutant p53 and mdm2 were significantly increased(P<0.01,P<0.05),while those ofα-globin,gata1 and hsp70 were obviously down-regulated(P<0.01,P<0.05,P<0.05).There were no significant differences between the rpl15 mutant and the control group in the mRNA expression levels of hematopoiesis-related transcription factors except erythroid-related ones.ConclusionThe rpl15 gene of zebrafish is successfully edited by CRISPR/Cas9 technique,and the mutant produces a phenotype similar to Diamoncl-Blackfan anemia disease.At the same time,it is suggested that p53 and hsp70 may be involved in regulating the formation of mutant phenotype.

关 键 词：CRISPR/Cas9 rpl15基因 DBA 斑马鱼 

分 类 号：R331.2[医药卫生—人体生理学] R394-33[医药卫生—基础医学]