Akgül Seçkin, Offenhäuser Carolin, Kordowski Anja, Day Bryan W
Sid Faithfull Brain Cancer Laboratory, Cell and Molecular Biology Department, QIMR Berghofer Medical Research Institute, Brisbane, QLD 4006, Australia.
School of Medicine and Dentistry, Griffith University, Gold Coast, QLD 4215, Australia.
Bioengineering (Basel). 2022 Feb 25;9(3):91. doi: 10.3390/bioengineering9030091.
Lentiviral vectors are unique and highly efficient genetic tools to incorporate genetic materials into the genome of a variety of cells whilst conserving biosafety. Their rapid acceptance made it necessary to improve existing protocols, including molecular engineering and cloning, production of purified lentiviral particles, and efficient infection of target cells. In addition to traditional protocols, which can be time-consuming, several biotechnology companies are providing scientists with commercially available lentiviral constructs and particles. However, these constructs are limited by their original form, tend to be costly, and lack the flexibility to re-engineer based on the ever-changing needs of scientific projects. Therefore, the current study organizes the existing methods and integrates them with novel ideas to establish a protocol that is simple and efficient to implement. In this study we, (i) generated an innovative site-directed nucleotide attachment/replacement and DNA insertion method using unique PCR primers, (ii) improved traditional methods by integrating plasmid clarification steps, (iii) utilized endogenous mRNA as a resource to construct new lentiviruses, and (iv) identified an existing purification method and incorporated it into an organized workflow to produce high-yield lentiviral particle collection. Finally, (v) we verified and demonstrated the functional validity of our methods using an infection strategy.
慢病毒载体是一种独特且高效的基因工具，可在确保生物安全性的同时将遗传物质整合到多种细胞的基因组中。它们迅速被广泛接受，这使得改进现有方案变得必要，这些方案包括分子工程和克隆、纯化慢病毒颗粒的生产以及对靶细胞的有效感染。除了可能耗时的传统方案外，几家生物技术公司正在为科学家提供商业上可获得的慢病毒构建体和颗粒。然而，这些构建体受到其原始形式的限制，往往成本高昂，并且缺乏根据科学项目不断变化的需求进行重新设计的灵活性。因此，当前的研究整理了现有方法，并将它们与新想法相结合，以建立一种易于实施的简单高效方案。在本研究中，我们：(i) 使用独特的PCR引物生成了一种创新的定点核苷酸连接/替换和DNA插入方法；(ii) 通过整合质粒澄清步骤改进了传统方法；(iii) 利用内源性mRNA作为资源构建新的慢病毒；(iv) 确定了一种现有的纯化方法，并将其纳入一个有组织的工作流程中，以产生高产的慢病毒颗粒收集物。最后，(v) 我们使用感染策略验证并证明了我们方法的功能有效性。
