AUTHOR=Sudarshan Gurucharan , Weil Simy , Rotem-Dai Noa , Manor Rivka , Greenshpan Yariv , Goldstein Oron , Sharabi Omri , Aflalo Eliahu D. , Ofir Rivka , Rosental Benyamin , Gazit Roi , Sagi Amir 

TITLE=Enhanced proliferation in a prawn embryonic primary cell culture ectopically expressing mutated Ras

JOURNAL=Frontiers in Marine Science

VOLUME=9

YEAR=2023

URL=https://www.frontiersin.org/journals/marine-science/articles/10.3389/fmars.2022.1100971

DOI=10.3389/fmars.2022.1100971

ISSN=2296-7745

ABSTRACT=<p>Crustacean cell line immortalization has gained a great deal of attention in recent decades for both scientific and applied reasons. Our goal in this study was to advance the state of art towards establishing an immortalized cell line by improving the proliferation rates of primary cells isolated from embryos of the giant freshwater prawn <italic>Macrobrachium rosenbergii</italic> by using a lentivirus expressing the <italic>Ras</italic> oncogene. The choice of <italic>Ras</italic> derived from its involvement in various cellular pathways, such as cell growth, differentiation, and survival, and its use as a tool for <italic>in-vitro</italic> immortalization, e.g., a specific mutated <italic>Ras</italic> (<italic>Ras<sup>V12</sup></italic>) was used to generate an arthropod cell line. Complementarily, <italic>in-silico</italic> screening of <italic>M. rosenbergii</italic> transcriptomic libraries for <italic>Ras</italic> expression indicated that <italic>Ras</italic> is already expressed at very early stages of embryo development. In the current study, we transduced primary <italic>M. rosenbergii</italic> embryonic cells with a lentivirus expressing <italic>Ras<sup>V12</sup></italic> by using the white spot syndrome virus (<italic>WSSV IE1</italic>) promoter. Expression and sequencing (as followed by sequencing cDNA, confocal microscopy and FACS analysis) of the mutated <italic>Ras</italic> in the transduced cells confirmed that the lentivirus was successfully integrated into the genome. The lenti<italic>-MrRas</italic> transduction rate was 23% in the total primary cell population and more than 80% in a sub-population of cells with high granularity. Proliferation of lenti-<italic>MrRas</italic> transfected cells was enhanced to almost 1200% of the seeding density by the end of our experiment (18 days), which was double that of the control. We were thus successful in enhancing the longevity of embryonic primary cell cultures by ectopic expression of the mutated <italic>Ras</italic> protein, but the improvement was not sufficient for immortalization.</p>