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The Amami rabbit (Pentagulus furnessi) is a dark brown-furred rabbit classified as an endangered species and only found in the Amami Islands of Japan. They are often called living fossils because they retain primitive characteristics of ancient rabbits that lived approximately 1 million years ago, such as short feet and hind legs and small ears. Although the ancient rabbit has disappeared due to the competition with European rabbit (Oryctolagus cuniculus) in the most of the Asian area, Amami rabbit survived since Amami Islands has isolated from Japan and Taiwan. Although Amari rabbit is one of the protected animals, their population decreases each year due to human activities, such as deforestation and roadkill. In this study, we collected roadkill samples of Amami rabbits and established primary and immortalized fibroblast cell lines. Combined expression of human-derived mutant Cyclin-dependent kinase 4, Cyclin D1, and hTERT allowed us to immortalize fibroblasts successfully in three individuals of Amami rabbits. The immortalized fibroblasts dramatically extended the cell culture period, when it was compared with the cell culture period of wild type cells. Furthermore, the immortalized cells maintained their normal chromosomal pattern (2n = 46). Our results suggest that cellular senescence which mainly regulated by p16-RB signaling pathway is conserved in animal evolution at least from 1 million years ago., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Isoleucyl-tRNA synthetase (IARS) syndrome is a recessive disease of Japanese Black cattle caused by a single nucleotide substitution. To repair the mutated IARS gene, we designed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to create a double-strand break near the mutation site. CRISPR/Cas9 and donor DNA that contained a synonymous codon for the correct amino acid and an Aequorea coerulescens Green Fluorescent Protein (AcGFP) cassette with a piggyBac transposase recognition site at both ends were introduced into bovine fetal fibroblast (BFF) cells isolated from a homozygous mutant calf. Recombinant cells were enriched on the basis of expression of AcGFP, and two cell lines that contained the repaired allele were subcloned. We generated somatic cell nuclear transfer (SCNT) embryos from the repaired cells and transferred 22 blastocysts to recipient cows. In total, five viable fetuses were retrieved at Days 34 and 36. PiggyBac transposase mRNA was introduced into BFF cells isolated from cloned foetuses and AcGFP-negative cells were used for second round of cloning. We transferred nine SCNT embryos to recipient cows and retrieved two fetuses at Day 34. Fetal genomic DNA analysis showed correct repair of the IARS mutation without any additional DNA footprint.

To obtain the data concerning death losses due to stillbirth, neonatal death and diseases in cloned cattle derived from somatic cell nuclear transfer (SCNT) and their progeny produced by Japanese institutions, a nationwide survey was carried out in July-August, 2006. As a result, lifetime data concerning 482 SCNT cattle (97.5% of cattle produced in the country at that time) and 202 progeny of SCNT cattle were accumulated and the death loss of these cattle was analyzed. Although 1/3 of delivered SCNT calves died during the perinatal period due to stillbirth and neonatal death, incidence of death loss due to diseases in SCNT cattle surviving more than 200 days after birth seems to be the same as these in conventionally bred cattle. In contrast, progeny of SCNT cattle showed the same level in death loss as observed in conventionally bred cattle throughout their lifetime. These results suggest that robust health would be expected in SCNT cattle surviving to adulthood and their progeny.

We investigated the possibility of producing calves from transferable bovine embryos obtained by nuclear transfer using somatic cell-derived cell lines. Muscle cells obtained from 2 Japanese Black bulls were dispersed in Hank's solution supplemented with collagenase Type-I. The separated muscle cells were cultured in Dulbecco's Modified Eagle's medium (D-MEM) supplemented with 10% fetal bovine serum (FBS) at 39 degrees C in an atmosphere of 5% CO2 in air. Cells were passaged at least 4 times, and for 5 d prior to nuclear transfer they (donor cells: karyoplasts) were cultured in D-MEM supplemented with 0.5% FBS (to induce quiescence) or 10% FBS. Recipient oocytes were produced by in vitro culture of bovine oocytes that were obtained at a slaughterhouse and then enucleated in modified phosphate buffered saline supplemented with cytochalasin B. Embryos were reconstructed by 3 protocols using karyoplasts cultured in the medium with 0.5% FBS. 1) Group A: recipient oocytes (cytoplasts; n = 157) were treated with Ca ionophore A 23187, ethanol and cycloheximide, and then a karyoplast was fused to an activated cytoplast. 2) Group B: karyoplasts were transferred to cytoplasts (n = 117), and the couplets were treated with electric stimulation and then Ca ionophore A 23187 and cycloheximide. 3) Group C: cytoplasts (n = 104) were cultured for a further 12 h before fusion, and then the couplets were treated with electric stimulation and cycloheximide. 4) Group D: in addition to the above 3 groups, karyoplasts cultured in the medium with 10% FBS were transferred to recipient cytoplasts (n = 137) and treated as in Protocol 2. Reconstructed embryos were cultured in modified CR1aa for 8 d, and the development of embryos was assessed. In total 73 blastocysts were obtained, and the frequency of development to the blastocyst stage in Group A (2.5%) was lower than that of Groups B, C and D (20.5, 18.3 and 19.0%, respectively; P < 0.01). Of these the sex of 21 blastocysts was determined by rapid Y-chromosome detection assay, and all were male, suggesting that nuclear replacement had been achieved successfully. When 26 blastocysts were transferred to 20 recipient cows, 8 of them became pregnant; 4 cows subsequently aborted about 60 d after embryo transfer while the remaining 4 cows calved. These results indicate that reconstructed embryos obtained by nuclear transfer of muscle cell-derived cell lines can develop to the blastocyst stage, and some are sufficiently competent to develop to term. Particularly important was the finding that special culture protocols for somatic cells prior to nuclear transfer were not necessary in our system.