Investigation of essential cell cycle regulator genes as candidates for immortalized shrimp cell line establishment based on the effect of in vitro culturing on gene expression of shrimp primary cells

Continuous shrimp cell cultures offer opportunities for studying shrimp pathogens at the cellular and molecular level and developing diagnostic tools. However, no continuous shrimp cell lines have yet been successfully established. This might be due to the lack of information on the molecular mechanisms that control shrimp cell proliferation and cell arrest under culture conditions. In this study, differentially expressed genes (DEGs) in shrimp primary culture cells were determined by comparison with normal tissue (in vivo). This study aimed to comprehensively identify key regulator genes that control cell proliferation and cell cycle arrest in culture conditions. The primary shrimp cells were derived from testicular tissue and cultured in vitro. RNA sequencing (RNA-Seq) was performed to investigate change in gene expression level across the entire transcriptome between shrimp primary cells and normal tissue (in vivo). RNA-seq results revealed over 100 genes with distinct gene expression patterns between primary cells and normal tissue. The DEG results and functional gene analysis showed a clear difference in gene expression patterns of cell cycle-related genes between primary cells and normal tissue. LRWD1, TMEM127, CDCA3, PPP2R1A, and GOLGA2, which are cell cycle-related genes, were downregulated in shrimp primary cells. These genes are required for G1/S phase transition, entry into mitosis, and cell proliferation control. However, a gene that induces cell cycle arrest, ARAF, was upregulated in culture conditions. The results demonstrated that these genes might play essential roles in cell proliferation and cell arrest under culture conditions. These candidate genes may be alternative potential targets for genetic manipulation to maintain cell proliferation and establishment of a shrimp continuous cell line.