INCREASING TRANSDUCTION EFFICIENCY IN MAMMALS: DEVELOPMENT OF A STABLE SF9 INSECT CELL LINE FOR THE PRODUCTION OF G-VSV PSEUDOTYPED BACULOVIRUSES

PLASTINE MARÍA DEL PILAR; AMALFI SABRINA; LÓPEZ MARÍA GABRIELA; OSCAR TABOGA; ALFONSO VICTORIA

Santa Fe

Simposio; IX Simposio Latinoamericano de Tecnología de Cultivos Celulares; 2022

Universidad Nacional del Litoral

Baculoviruses are promising vectors for gene delivery in mammals, although they exhibit distinct levels of transduction according to the cell line. Pseudotyping viral vectors is a widely used strategy to enhance cell tropism and increase transduction efficiency. In this work, we developed a stable Sf9 insect cell line that expresses the G-protein of the vesicular stomatitis virus (G-VSV) under the very late strong and infection-inducible promoter pXXL, previously constructed in our laboratory. First, we cloned the G-VSV gene in a plasmid carrying a blasticidin resistance gene and we transfected it into Sf9 cells. The recombinant cells were selected by antibiotic resistance and diluted twice to obtain an oligoclonal cell line. Due to G-VSV is a fusogenic protein, we selected some clones based on the size and number of syncytia obtained after infection with baculovirus. Then, we evaluated the level of G-VSV expression by immunodetection in cell extracts after infection. Finally, we selected the cells that showed the highest incorporation of G-VSV in viral particles. To determine the infection conditions for the best cell-line performance, we obtained a baculovirus that expresses eGFP in mammals using different multiplicities of infection and harvest times and then measured the reporter gene expression by fluorescence microscopy and flow cytometry in mammalian cells. Using different pseudotyped baculoviruses, a consistent increase in the transduction efficiency was detected in all the tested mammalian cell lines. The results highlight the feasibility and convenience of improving the performance of gene delivery without inserting the pseudotyping gene into the baculoviral genomes.