209 CLONED EMBRYONIC DEVELOPMENT AND GENE EXPRESSION OF SPONTANEOUSLY IMMORTALIZED PORCINE SKIN FIBROBLASTS

Abstract

Immortalization of somatic cells by oncogenes is effective for evaluation of gene targeting tools or molecular cell pathway because of relatively longer life span than normal cells. The BMI1 gene has been used to immortalize human and murine somatic cells, and we confirmed that BMI1 increased cell-life span of porcine skin fibroblasts and had no detrimental effect on pre-implantational development after somatic-cell nuclear transfer (SCNT; unpublished data). Here, we report a primary cell-line which was spontaneously immortalized without transduction of any additional gene. The minipig skin fibroblasts (passage 3 after primary culture) were divided into two parts, one group was electroporetically transfected with pCAG-BMI1-T2A-RFP plasmids (BC1) and the other had no treatment (CC1). To establish the single-cell-originated cell-lines, cells of each group were plated in 100-mm dishes at 100 cells/dish and well-formed colonies were picked up after 2 weeks. These colonies were expanded until they were fully confluent in 100-mm dishes (designated Passage 0) and then, the cell were maintained with DMEM (20% FBS) at 3 × 10⁵ cells/60 mm dish. Population doubling time was checked every 5 passages until Passage 45 (sub-cultured during ~160 days) by calculation of cells that had been plated in 12-well plates at 4 × 10⁴ cells/well. The expressions of p16, p21, and DNMT3b genes were determined by RT-qPCR at early (Passage 5) and late (Passage 45) stage. Also, SCNT (Song et al. 2009 Mol. Reprod. Dev. 76, 611–619) embryos using cells of early and middle (Passage 35) stage were evaluated in terms of reprograming efficiency. Data were analysed by t-test (Prism version 5.01, GraphPad Software, La Jolla, CA, USA). While the mean doubling time of BC1 was 25.3 h until Passage 40 and drastically increased at Passage 45 (81.7 h), that of CC1 was maintained until Passage 45 (mean 38.3 h). Expression of p16 in CC1 was significantly higher than that in BC1 at all stages. However, in late stage CC1, p21 expression was significantly lower than other groups and DNMT3b expression was increased. In SCNT embryos, the rate of blastocysts with early stage CC1 (18.3%) was not different from that of early stage BC1 (19.9%). And, although the rates of SCNT blastocyst derived from middle stage cells were decreased than those of early stage cells, there was no difference between BC1 and CC1 (6.6% and 5.4%, respectively). In karyotyping, while BC1 was trisomy of chromosome 14 only in late stage, CC1 had an isochromosome in chromosome 17 from early stage and an additional part was attached in chromosome 11 at late stage CC1. In summary, spontaneously immortalized skin fibroblasts could maintain the cell-life span by down-regulating the p21 expression and the pre-implantational development after SCNT was not different from that of BMI1-immortalized cells. And, additional studies are needed to confirm whether the chromosomal abnormality influences the expression of other genes related with cell cycle or senescence.

This study was supported by NRF-2013R1A1A2010766, IPET (#311011-05-3-SB010), the Research Institute for Veterinary Science, and the BK21 plus program.