Key Laboratory of Marine Genetics and Breeding, Ministry of Education, and College of Marine Life Sciences, Ocean University of China, Qingdao, 266003, China.
Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao, 266003, China.
In Vitro Cell Dev Biol Anim. 2021 Oct;57(9):912-931. doi: 10.1007/s11626-021-00622-1. Epub 2021 Nov 1.
A new continuous fish cell line (CAM) has been successfully derived from the muscle tissues of grass goldfish, Carassius auratus. The primary cell cultures were initiated by incomplete trypsinization first and then explant culture in a Leibovitz-15 medium supplemented with 15% fetal bovine serum and 10% fish muscle extract. It was found that the CAM cells were very sensitive to trypsinization and needed to be sub-cultured at a low trypsin concentration of 0.0625% to be able to go through the crisis of spontaneous immortalization transformation, and afterward a total of five derivative cell strains were isolated from the original CAM cell line. This spontaneous immortalization transformation event was recorded successively at passages 44-47, beginning with a large-scale apoptosis and senescence and followed by mitosis arrest and re-activation, thus designated as cell strain CAM-44A, 44B, 45A, 44B, and 47A. Now both the CAM cell line and strains had been sub-cultured for more than 89 times and could be well cryopreserved in the growth medium containing 5% dimethylsulfoxide. Chromosome analysis and COI gene analysis had confirmed the grass goldfish origin of these CAM cells. Transfection potential analysis indicated that Lipofectamine LTX and Xfect were two suitable transfection reagents to be used in the gene delivery of CAM cells with a transfection efficiencies up to 11±6% and 8±3% in the CAM cell lines, respectively. Among the five cell strains, CAM-47A showed the highest transfection potential with a transfection efficiency up to 28 ± 5%. This work will provide a useful cell source for works on the cell-based artificial fish meat production and functional studies of fish myogenesis-related genes.
一种新的草鱼（Carassius auratus）连续细胞系（CAM）已成功地从草鱼的肌肉组织中衍生而来。原代细胞培养首先通过不完全胰蛋白酶消化进行，然后在补充有 15%胎牛血清和 10%鱼肌肉提取物的 Leibovitz-15 培养基中进行组织块培养。结果发现，CAM 细胞对胰蛋白酶非常敏感，需要在低浓度（0.0625%）的胰蛋白酶下进行传代，才能通过自发永生化转化的危机，之后从原 CAM 细胞系中分离出 5 株衍生细胞系。该自发永生化转化事件在第 44-47 代中被连续记录，首先是大规模的细胞凋亡和衰老，随后是有丝分裂停滞和再激活，因此被命名为细胞系 CAM-44A、44B、45A、44B 和 47A。现在，CAM 细胞系和细胞株已传代 89 多次以上，并能在含 5%二甲基亚砜的生长培养基中良好冻存。染色体分析和 COI 基因分析证实了这些 CAM 细胞来源于草鱼。转染潜能分析表明，Lipofectamine LTX 和 Xfect 是两种适合于 CAM 细胞基因转染的转染试剂，转染效率分别可达 11±6%和 8±3%。在这 5 株细胞系中，CAM-47A 的转染潜能最高，可达 28 ± 5%。这项工作将为基于细胞的人工鱼肉生产和鱼类肌肉发生相关基因的功能研究提供有用的细胞来源。
