Cellular conservation of endangered midget buffalo (Lowland Anoa, Bubalus quarlesi) by establishment of primary cultured cell, and its immortalization with expression of cell cycle regulators

Abstract

Lowland Anoa has become endangered due to hunting and human activity. Protection and breeding of endangered species in a controlled environment is the best way of conservation. However, it is not possible to adopt this approach for all endangered species because of the cost involved and the ever-increasing number of critically endangered species. In consideration of these limitations to the conventional conservation methods, we established a primary cell culture of endangered buffalo (Lowland Anoa, Bubalus quarlesi), for the preservation of this biological resource. In addition, we introduced human derived, mutant cyclin dependent kinase 4 (CDK4), Cyclin D, and telomerase reverse transcriptase (TERT) into the primary cells. The successful introduction of these three genes was confirmed by western blot with specific antibodies, and enzymatic activity. We also showed that the expression of mutant CDK4, Cyclin D, and TERT allows us to efficiently establish an immortalized cell line, with an intact chromosome pattern, from Lowland Anoa. To the best of our knowledge, this study is the first investigation that established an immortalized cell line of an endangered wild animal species.

Keywords: Cellular conservation; Cultured cells; Endangered animals; Immortalization.

Fig. 1

Detection of potential morphological changes and fluorescence of Lowland Anoa derived cells following gene introduction using recombinant viral vectors. a Cell morphology of 4 cell lines; wild type; EGFP, cell line transduced with EGFP expressing virus, K4D, cell line transduced with mutant CDK4 and Cyclin D; K4DT, cell line transduced with cyclin D1, mutant CDK4 and TERT. b Detection of fluorescence in cells transduced with the recombinant lentiviral vector expressing enhanced green fluorescence protein (EGFP). Note that the fluorescence gene was introduced into the cells around 50 %. The morphologies of cells were obtained after 72 h from transduction with recombinant viral vectors

Fig. 2

Biological characterization of the cell line established from recombinant cells of Lowland Anoa. a Genomic PCR analysis of Cyclin D1, mutant CDK4, TERT and the internal control (NADH dehydrogenase subunit 1). b Western blot of Cyclin D1, mutant CDK4 and tubulin. c Stretch PCR assay for the detection of telomerase activity. Note that K4DT cells were positive for the telomerase activity, while the negative control, wild type, EGFP, and K4D were not. The DNAs from passage 17 were analyzed

Fig. 3

Cell cycle analysis in wild type and K4DT cell lines derived from Lowland Anoa. a Cell cycle analysis of wild type Lowland Anoa derived cells. b Cell cycle analysis of EGFP expressing Lowland Anoa derived cells. c Cell cycle analysis of K4D cells. d Cell cycle analysis of K4DT cells. The cell cycle at passage 17 was analyzed

Fig. 4

Karyotype analysis in wild type and K4DT cells. a Kayotype analysis of wild type Lowland Anoa derived cells. b Karyotype analysis of K4DT cells, indicating that K4DT cells retained intact original pattern (2n = 48) of Lowland Anoa. Representative images are shown. The chromosome patterns around passage 12 are shown

Fig. 5

Population doubling assay and SA-β-Gal staining of wild type and Lowland Anoa derived recombinant cells. a Population doubling assay in 4 cell lines; WT, EGFP, K4D, and K4DT cell lines. K4DT cells showed continued cell proliferation beyond passage 26, while the wild type and EGFP transfected cells ceased to divide at around PD value of 60. b The result of SA-β-Gal staining in wild type, EGFP expressing cells (EGFP), K4D and K4DT cell lines at passage 24. Note that all of the wild type, EGFP, and K4D cells showed positive blue staining, while the K4DT cells were negative for SA-β-Gal staining, and maintained small cell size

Fig. 6

Cell cycle analysis after the cell synchronization with Nocodazole and Aphidicolin. a Representative results of 2 h from release. b Results of 7 h from release. c Results of 14 h from release. d Results of 24 h from release. Wild type and K4DT cells were synchronized into G1 phase with Nocodazole and Aphidicolin treatment. Note that K4DT showed good cell synchronization, while wild type did not. No synchronization means that these cells were not treated with Nocodazole and Aphidicolin

Fig. 7

Amino acid sequence alignment of human and water buffalo derived cyclin dependent kinase 4. The conserved region is highlighted in black. The activation loop domain is underlined