Engineering Novel Lentiviral Vectors for Labelling Tumour Cells and Oncogenic Proteins
Seçkin Akgül; Carolin Offenhäuser; Anja Kordowski; Bryan W. Day
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.
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