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)

Abstract

Conservation of the genetic resources of endangered animals is crucial for future generations. The loggerhead sea turtle (Caretta caretta) is a critically endangered species, because of human hunting, hybridisation with other sea turtle species, and infectious diseases. In the present study, we established primary fibroblast cell lines from the loggerhead sea turtle, and showed its species specific chromosome number is 2n = 56, which is identical to that of the hawksbill and olive ridley sea turtles. We first showed that intensive hybridization among multiple sea turtle species caused due to the identical chromosome number, which allows existence of stable hybridization among the multiple sea turtle species. Expressions of human-derived mutant Cyclin-dependent kinase 4 (CDK4) and Cyclin D dramatically extended the cell culture period, when it was compared with the cell culture period of wild type cells. The recombinant fibroblast cell lines maintained the normal chromosome condition and morphology, indicating that, at the G1/S phase, the machinery to control the cellular proliferation is evolutionally conserved among various vertebrates. To our knowledge, this study is the first to demonstrate the functional conservation to overcome the negative feedback system to limit the turn over of the cell cycle between mammalian and reptiles. Our cell culture method will enable the sharing of cells from critically endangered animals as research materials.

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

“…The combined expression of mutant CDK4, Cyclin D1, and TERT enables us to efficiently immortalize various cell types, as mentioned previously. The basic principle of this K4DT immortalization method is bypassing the negative feedback signal from the senescence protein p16 (Fukuda et al, 2018). Once cells reach the senescence phase, p16 accumulates in the cells (Shiomi et al, 2011).…”

“…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). Cells immortalized using this method maintain the cell differentiation and chromosome patterns of the original cells (Shiomi et al, 2011).…”

“…The packaging of retrovirus was carried out in 293T cell under the transient expression of QCXIN-AR or QCXIN-EGFP, and packaging plasmids, pCL-gag pol, and pCMV-VSV-G-RSV-Rev (Dr. Hiroyuki Miyoshi, RIKEN BioResorce Center, Tsukuba, Japan). The detailed packaging procedure was described in our previous report (Fukuda et al, 2018). We named immortalized DPCs with androgen receptor expression K4DT-AR, and immortalized DPCs with control EGFP expression K4DT-EGFP.…”

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

“…This immortalization method using mutant CDK4, Cyclin D1, and TERT was termed "K4DT" in reference to the introduced genes. The chromosomal pattern of cells established using the K4DT method is retained, along with the nature of primary cells, possibly due to the intact function of p53 [13,[15][16][17]. We also recently demonstrated that our corneal epithelial cell line, established with the K4DT immortalization method, can be a useful tool to detect eye toxicity, and it can be used as a new resource for in vitro ocular toxicity testing [18].…”

“…To immortalize primary human dental pulp stem cells, we prepared recombinant retroviruses expressing R24C mutant cyclin-dependent kinase 4 (CDK4 R24C ), Cyclin D1, and TERT. PQCXIP-CDK4R24C (puromycin-resistant), pQCXIN-Cyclin D1 (G418-resistant), and pCLXSH-TERT (hygromycin B-resistant) retroviral plasmids as well as pQCXIN-EGFP (G418-resistant) as a control expressing EGFP to monitor the efficiency of infection were constructed as described previously [16]. These retroviral plasmids were co-transfected into 293T cells together with packaging plasmids, pCL-GagPol and pCMV-VSV-G-RSV-Rev, by using the lipofection method [19].…”