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. 2018 Dec;70(6):1619-1630.
doi: 10.1007/s10616-018-0254-0. Epub 2018 Sep 17.

Efficient immortalization of cells derived from critically endangered Tsushima leopard cat (Prionailurus bengalensis euptilurus) with expression of mutant CDK4, Cyclin D1, and telomerase reverse transcriptase

Affiliations

Efficient immortalization of cells derived from critically endangered Tsushima leopard cat (Prionailurus bengalensis euptilurus) with expression of mutant CDK4, Cyclin D1, and telomerase reverse transcriptase

Ryo Gouko et al. Cytotechnology. 2018 Dec.

Abstract

Tsushima leopard cat is the subspecies of Amur cats, and it is classified as the most highest class of critically endangered animals. Although the protection activity is highly recognized, the number of animals is decreasing due to the human activity and invasion of domestic cats and infectious disease. In this study, we succeeded primary culture of normal fibroblasts derived from the Tsushima leopard cat (Prionailurus bengalensis euptilurus). Furthermore, we introduced the human derived mutant Cyclin Dependent Kinase 4, Cyclin D1, and telomere reverse transcriptase. We showed that the expression of these three genes efficiently immortalized cells derived from Tsushima leopard cat. Furthermore, we showed that the chromosome pattern of the established cells is identical with the original one. These data indicate that our method of immortalization is useful to establish cell lines from critically endangered cats, which potentially contributes to the re-generation of critically endangered animals from cultured cell with reproductive technique, such as somatic cloning.

Keywords: Cellular senescence; Endangered animals; Immortalization; Tsushima leopard cat.

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Figures

Fig. 1
Fig. 1
Appearance and cell morphology, gene introduction into Tsushima leopard cat derived cells. a Appearance of Tsushima leopard cat. b The cell morphology of the Tsushima leopard cat derived cells. c Fluorescence in cells transduced with the recombinant lentivirus expressing enhanced green fluorescence protein (EGFP). CS-CMV-EGFP (upper panel); control (lower panel)
Fig. 2
Fig. 2
The detection of genomic cassette by genomic PCR and the protein expression by western blotting, the telomerase activity by Stretch PCR. a The detection of genomic cassette. Lane1; parental cells, Lane2; K4D, Lane3; K4DT + T. The amplification products from the expression cassette of Cyclin D (Upper left), CDK4 (Lower left), TERT (Upper right), and internal control (Lower right, TSC2, Tsushima leopard cat derived Tuberous Sclerosis Type II gene). b The detection of protein expression. Lane1; K4DT + T, Lane2; K4D, Lane3; parental cells. c The detection of the telomerase activity by stretch PCR
Fig. 3
Fig. 3
Growth curve and SA-beta-Gal staining of the Tsushima leopard cat derived cells and the immortalized cells. a Analysis of the cell proliferation. The result shows the each accumulated population doubling of parental, K4D and K4DT + T cells through sequential passaging. b Detection of senescent cells of parental, K4D and K4DT + T cells at passage 2 by SA-beta-Gal staining. Upper panels, staining features with lower magnification. Lower panels, staining features with higher magnification
Fig. 4
Fig. 4
Cell cycle analysis of parental, K4D and K4DT + T cells at different passages. a Parental cells, b K4D cells, c K4DT + T cells at passage 2. d K4D cell, E. K4DT + T cells at passage 7. The representative data of each experimental group were listed. The mean and standard deviation of the ratio of each cell cycle stage from six samples are shown in Table 1
Fig. 5
Fig. 5
Karyotype analysis of Tsushima leopard cat derived K4DT + T cells. a G-banding metaphase spread of K4DT + T cell. b Aligned chromosome of Tsushima leopard cat derived K4DT + T cell. Sex chromosomes are indicated as X and Y

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