RNA interference （RNAi） refers to the process of exogenous double-stranded RNA （dsRNA） silencing the complementary endogenous messenger RNA. RNAi has been widely used in entomological research for functional genom...RNA interference （RNAi） refers to the process of exogenous double-stranded RNA （dsRNA） silencing the complementary endogenous messenger RNA. RNAi has been widely used in entomological research for functional genomics in a variety of insects and its potential for RNAi-based pest control has been increasingly emphasized mainly because of its high specificity. This review focuses on the approaches of introducing dsRNA into insect cells or insect bodies to induce effective RNAi. The three most common delivery methods, namely, microinjection, ingestion, and soaking, are illustrated in details and their advantages and limitations are summarized for purpose of feasible RNAi research. In this review, we also briefly introduce the two possible dsRNA uptake machineries, other dsRNA delivery methods and the history of RNAi in entomology. Factors that influence the specificity and efficiency of RNAi such as transfection reagents, selection of dsRNA region, length, and stability of dsRNA in RNAi research are discussed for further studies.展开更多
Custom-designed nuclease technologies such as the clustered regularly in- terspaced short palindromic repeat （CRISPR）-associated （Cas） system provide attractive genome editing tools for insect functional genetics....Custom-designed nuclease technologies such as the clustered regularly in- terspaced short palindromic repeat （CRISPR）-associated （Cas） system provide attractive genome editing tools for insect functional genetics. The targeted gene mutagenesis me- diated by the CR1SPR/Cas9 system has been achieved in several insect orders including Diptera, Lepidoptera and Coleoptera. However, little success has been reported in agricul- tural pests due to the lack of genomic information and embryonic microinjection techniques in these insect species. Here we report that the CRISPR/Cas9 system induced efficient gene mutagenesis in an important Lepidopteran pest Spodoptera litura. We targeted the S. litura Abdominal-A （Slabd-A） gene which is an important embryonic development gene and plays a significant role in determining the identities of the abdominal segments of in- sects. Direct injection of Cas9 messenger RNA and Slabd-A-specific single guide RNA （sgRNA） into S. litura embryos successfully induced the typical abd-A deficient pheno- type, which shows anomalous segmentation and ectopic pigmentation during the larval stage. A polymerase chain reaction-based analysis revealed that the Cas9/sgRNA complex effectively induced a targeted mutagenesis in S. litura. These results demonstrate that the CRISPR/Cas9 system is a powerful tool for genome manipulation in Lepidopteran pests such as S. litura.展开更多
加州大学医学院 Teh Ping Lin 博士早在1966年即利用小鼠受精卵原核显微注射技术,系统地研究了微注射不同量外源物质(石蜡油、牛γ-球蛋白溶液)对小鼠胚胎受体移植后的分裂、发育的影响程度,并预见该技术用于研究小鼠或其它种类哺乳动...加州大学医学院 Teh Ping Lin 博士早在1966年即利用小鼠受精卵原核显微注射技术,系统地研究了微注射不同量外源物质(石蜡油、牛γ-球蛋白溶液)对小鼠胚胎受体移植后的分裂、发育的影响程度,并预见该技术用于研究小鼠或其它种类哺乳动物胚胎分化及遗传效应具有潜在价值。直到1980年,随着分子生物学技术的迅速发展,Gordon 等人首先采用显微注射法育成带有人胸苷激酶基因这一外源 DNA 片断的'新型'转基因小鼠后。展开更多
文摘RNA interference （RNAi） refers to the process of exogenous double-stranded RNA （dsRNA） silencing the complementary endogenous messenger RNA. RNAi has been widely used in entomological research for functional genomics in a variety of insects and its potential for RNAi-based pest control has been increasingly emphasized mainly because of its high specificity. This review focuses on the approaches of introducing dsRNA into insect cells or insect bodies to induce effective RNAi. The three most common delivery methods, namely, microinjection, ingestion, and soaking, are illustrated in details and their advantages and limitations are summarized for purpose of feasible RNAi research. In this review, we also briefly introduce the two possible dsRNA uptake machineries, other dsRNA delivery methods and the history of RNAi in entomology. Factors that influence the specificity and efficiency of RNAi such as transfection reagents, selection of dsRNA region, length, and stability of dsRNA in RNAi research are discussed for further studies.
基金Acknowledgments This work was supported by grants from the Strategic Priority Research Program of Chinese Academy of Sciences （XDB11010500）, the External Cooperation Program of BIC, Chinese Academy of Sciences （GJHZ201305） and the National Natural Science Foundation of China （31420103918 and 31372257）.
文摘Custom-designed nuclease technologies such as the clustered regularly in- terspaced short palindromic repeat （CRISPR）-associated （Cas） system provide attractive genome editing tools for insect functional genetics. The targeted gene mutagenesis me- diated by the CR1SPR/Cas9 system has been achieved in several insect orders including Diptera, Lepidoptera and Coleoptera. However, little success has been reported in agricul- tural pests due to the lack of genomic information and embryonic microinjection techniques in these insect species. Here we report that the CRISPR/Cas9 system induced efficient gene mutagenesis in an important Lepidopteran pest Spodoptera litura. We targeted the S. litura Abdominal-A （Slabd-A） gene which is an important embryonic development gene and plays a significant role in determining the identities of the abdominal segments of in- sects. Direct injection of Cas9 messenger RNA and Slabd-A-specific single guide RNA （sgRNA） into S. litura embryos successfully induced the typical abd-A deficient pheno- type, which shows anomalous segmentation and ectopic pigmentation during the larval stage. A polymerase chain reaction-based analysis revealed that the Cas9/sgRNA complex effectively induced a targeted mutagenesis in S. litura. These results demonstrate that the CRISPR/Cas9 system is a powerful tool for genome manipulation in Lepidopteran pests such as S. litura.
文摘加州大学医学院 Teh Ping Lin 博士早在1966年即利用小鼠受精卵原核显微注射技术,系统地研究了微注射不同量外源物质(石蜡油、牛γ-球蛋白溶液)对小鼠胚胎受体移植后的分裂、发育的影响程度,并预见该技术用于研究小鼠或其它种类哺乳动物胚胎分化及遗传效应具有潜在价值。直到1980年,随着分子生物学技术的迅速发展,Gordon 等人首先采用显微注射法育成带有人胸苷激酶基因这一外源 DNA 片断的'新型'转基因小鼠后。
基金This work was supported by the National 973 Program (No.2001CB109006), the Scientific Funds of the Educational Bureau of Hunan Province (No.04C378), and the Ph.D Programs Foundation in Hunan Normal University (No. 24030610)
