Establishment of an immortalized cell line derived from the prairie vole via lentivirus-mediated transduction of mutant cyclin-dependent kinase 4, cyclin D, and telomerase reverse transcriptase

Abstract

The prairie vole (Microtus ochrogaster) shows social behaviors such as monogamy and parenting of infants with pair bonding. These social behaviors are specific to the prairie vole and have not been observed in other types of voles, such as mountain voles. Although the prairie vole has several unique characteristics, an in vitro cell culture system has not been established for this species. Furthermore, establishment of cultured cells derived from the prairie vole may be beneficial based on the three Rs (i.e., Replacement, Reduction, and Refinement) concept. Therefore, in this study, we attempted to establish an immortalized cell line derived from the prairie vole. Our previous research has shown that transduction with mutant forms of cyclin-dependent kinase 4 (CDK4), cyclin D, and telomerase reverse transcriptase (TERT) could efficiently immortalize cells from multiple species, including humans, cattle, pigs, and monkeys. Here, we introduced these three genes into prairie vole-derived muscle fibroblasts. The expression of mutant CDK4 and cyclin D proteins was confirmed by western blotting, and telomerase activity was detected in immortalized vole muscle-derived fibroblasts (VMF-K4DT cells or VMFs) by stretch PCR. Population doubling analysis showed that the introduction of mutant CDK4, cyclin D, and TERT extended the lifespan of VMFs. To the best of our knowledge, this is the first report describing the establishment of an immortalized cell line derived from the prairie vole through the expression of mutant CDK4, cyclin D, and human TERT.

Human Derived Immortalized Dermal Papilla Cells With a Constant Expression of Testosterone Receptor

“…The addition of TERT expression allows for extension of the telomere sequence, resulting in immortalization. We previously reported that this K4DT method can be applied to various cell types, such as fibroblasts (Katayama et al, 2016) and intestinal epithelial cells (Kuroda et al, 2015b). In this study, we showed that expression of mutant CDK4, Cyclin D, and TERT allows us to efficiently establish immortalized cells from human DPCs.…”

“…Our research group previously reported that combined expression of R24C mutant cyclin-dependent kinase 4 (CDK4), Cyclin D1, and telomere reverse transcriptase (TERT) allowed us to efficiently immortalize human- (Shiomi et al, 2011), cattle and pig- (Donai et al, 2014), prairie vole- (Katayama et al, 2016(Katayama et al, , 2017, monkey- (Kuroda et al, 2015a), midget buffalo- (Fukuda et al, 2016), and mega bat- (Tani et al, 2019), Tsushima wildcat-derived cells (Gouko et al, 2018). Furthermore, growth acceleration with mutant CDK4 and Cyclin D1 is conserved even in sea turtles, suggesting that the underlying cell cycle mechanism was well-conserved throughout animal evolution (Fukuda et al, 2018).…”

Expression of human mutant cyclin dependent kinase 4, Cyclin D and telomerase extends the life span but does not immortalize fibroblasts derived from loggerhead sea turtle (Caretta caretta)

“…showed that immortalised human myogenic cells maintained their original nature as myogenic stem cells, with the intact chromosome condition 10 . We previously demonstrated the immortalisation of fibroblast cell lines derived from bovine, porcine 9 and non-human primate species 11 , midget buffalo 14 , prairie vole 12 , 13 , 32 , bovine colon epithelial cell 8 by transgene expression of human mutant CDK4, Cyclin D and TERT. The success of the immortalisation relies on the conserved homology of the cell cycle regulators, CDK4 and Cyclin D, over multiple species.…”

“…Furthermore, our research group previously reported that expression of human-derived mutant Cyclin-dependent kinase 4 (CDK4), Cyclin D and Telomerase Reverse Transcriptase (TERT) efficiently immortalises cells of various mammalian species, including human, bovine 8 , swine 9 , 10 , monkey 11 , prairie vole 12 , 13 and midget buffalo 14 . We designated this recently developed method as K4DT, based on the identities of the introduced genes (mutant CD K4 , Cyclin D and T ERT).…”

Efficient immortalization of human dental pulp stem cells with expression of cell cycle regulators with the intact chromosomal condition

“…Our research group has previously reported efficient immortalization in multiple species via co-expression of R24C mutant cyclin-dependent kinase 4 (CDK4 R24C ), Cyclin D1, and telomere reverse transcriptase (TERT) [8][9][10][11][12][13][14]. This immortalization method using mutant CDK4, Cyclin D1, and TERT was termed "K4DT" in reference to the introduced genes.…”