报道表达重组病毒是一个很好的选择和强大的工具,其中,病毒感染,致病性,和传输,以及确定抑制病毒感染和预防性疫苗的治疗化合物。为了对抗正在进行的2019年冠状病毒病(COVID-19)大流行,我们建立了一个强大的基于细菌人工染色体(BAC)的反向遗传学(RG)系统,以快速产生重组严重急性呼吸综合征冠状病毒2(rSARS-CoV-2),从而研究病毒蛋白在病毒发病机制中的作用.此外,我们已经设计了表达报告基因的重组病毒,其中我们将报告基因置于病毒核衣壳(N)基因的上游,以促进高水平的报告基因表达,这有利于SARS-CoV-2的体外和体内研究。迄今为止,我们已经与全球100多个实验室共享了基于BAC的RG系统,这有助于加快SARS-CoV-2的调查。然而,包含整个SARS-CoV-2基因组(〜30,000nt)的BAC的遗传操作具有挑战性。在这里,我们使用基于BAC的RG方法为工程rSARS-CoV-2提供技术细节。我们描述了(i)将全长(FL)SARS-CoV-2基因组序列组装到空pBeloBAC中,(ii)pBeloBAC-FL的验证,(iii)将金星报告基因克隆到pBeloBAC-FL中,和(iv)表达Venus的rSARS-CoV-2的回收。通过遵循这个协议,具有基本分子生物学和基因工程技术知识的研究人员将能够产生野生型(WT)和报告表达rSARS-CoV-2。重要性我们已经建立了基于细菌人工染色体(BAC)的RG系统,以产生重组的严重急性呼吸综合征冠状病毒2(rSARS-CoV-2),并设计了表达报告基因的重组病毒,以评估体外和体内的病毒感染。迄今为止,我们已经与全球100多个实验室共享了基于BAC的RG系统,这有助于加快SARS-CoV-2的调查。然而,含有约30,000个核苷酸的全长SARS-CoV-2基因组的BAC的遗传操作具有挑战性。这里,我们提供了成功生成野生型(WT)重组SARS-CoV-2(rSARS-CoV-2)所需的所有详细实验步骤。同样,我们提供了有关如何从病毒核衣壳(N)蛋白基因座产生和挽救表达高水平金星荧光报告基因的rSARS-CoV-2的综合方案。通过遵循这些协议,具有分子生物学基础知识的研究人员将能够在40天内产生WT和表达Venus的rSARS-CoV-2。
Reporter-expressing recombinant virus represents an excellent option and a powerful tool to investigate, among others, viral infection, pathogenicity, and transmission, as well as to identify therapeutic compounds that inhibit viral infection and prophylactic vaccines. To combat the ongoing coronavirus disease 2019 (COVID-19) pandemic, we have established a robust bacterial artificial chromosome (BAC)-based reverse genetics (RG) system to rapidly generate recombinant severe acute respiratory syndrome coronavirus 2 (rSARS-CoV-2) to study the contribution of viral proteins in viral pathogenesis. In addition, we have engineered reporter-expressing recombinant viruses in which we placed the reporter genes upstream of the viral nucleocapsid (N) gene to promote high levels of reporter gene expression, which facilitates the study of SARS-CoV-2 in vitro and in vivo. To date, we have shared our BAC-based RG system with more than 100 laboratories around the world, which has helped to expedite investigations with SARS-CoV-2. However, genetic manipulation of the BAC containing the entire SARS-CoV-2 genome (~30,000 nt) is challenging. Herein, we provide the technical details to engineer rSARS-CoV-2 using the BAC-based RG approach. We describe (i) assembly of the full-length (FL) SARS-CoV-2 genome sequences into the empty pBeloBAC, (ii) verification of pBeloBAC-FL, (iii) cloning of a Venus reporter gene into pBeloBAC-FL, and (iv) recovery of the Venus-expressing rSARS-CoV-2. By following this protocol, researchers with knowledge of basic molecular biology and gene engineering techniques will be able to generate wild-type (WT) and reporter-expressing rSARS-CoV-2. IMPORTANCE We have established a bacterial artificial chromosome (BAC)-based RG system to generate recombinant severe acute respiratory syndrome coronavirus 2 (rSARS-CoV-2) and to engineer reporter-expressing recombinant viruses to assess viral infection in vitro and in vivo. To date, we have shared our BAC-based RG system with more than 100 laboratories around the world, which has helped to expedite investigations with SARS-CoV-2. However, genetic manipulation of the BAC containing the full-length SARS-CoV-2 genome of ~30,000 nucleotides is challenging. Here, we provide all the detailed experimental steps required for the successful generation of wild-type (WT) recombinant SARS-CoV-2 (rSARS-CoV-2). Likewise, we provide a comprehensive protocol on how to generate and rescue rSARS-CoV-2 expressing high levels of a Venus fluorescent reporter gene from the locus of the viral nucleocapsid (N) protein. By following these protocols, researchers with basic knowledge in molecular biology will be able to generate WT and Venus-expressing rSARS-CoV-2 within 40 days.