Primary fibroblast cultures and karyotype analysis for the olive ridley sea turtle (Lepidochelys olivacea)
Primary fibroblast cultures and karyotype analysis for the olive ridley sea turtle (Lepidochelys olivacea)
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橄榄龟（Lepidochelys olivacea）的原代成纤维细胞培养和核型分析
DOI：
10.1007/s11626-013-9715-0
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发表时间：
2014
期刊：
影响因子：
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通讯作者：
Isogai E. Inoue-Murayama M.
中科院分区：
文献类型：
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作者：
Fukuda T;Katayama M. Kinoshita K. Kasugai T;Okamoto H. Kobayashi K. Kurita M. Soichi M. Douai K. Uchida T;Onuma M;Sone H;Isogai E. Inoue-Murayama M.
由于人类活动，如渔业副渔获物（Peckham等人，2007年）、石油泄漏和海洋污染（Witherington，2001年），野生海龟的数量正在减少。此外，非法狩猎其油，肉和贝壳仍在继续（Koch等人，2013）。因此，各国都在实施保护措施，以保护濒临灭绝的海龟。在过去的几十年里，野生海龟的一个主要问题是杂交。种间杂交已在世界多个地区报道（Karl et al. 1995; Barber et al. 2003; Lara-Ruiz et al. 2006）。最初的海龟物种，如玳瑁、红海龟和橄榄龟，尽管它们的栖息地有重叠，但已经作为独立物种存在了数百万年（Bowen等人，1993年）。然而，最近关于这些物种之间密集杂交的报告清楚地表明，这些独特的原始物种在不久的将来面临灭绝的高风险。获取这些物种的基本遗传背景的信息，如核型，是必要的，以了解这种密集的杂交的原因。本研究建立了绿海龟的原代培养细胞，并对原代细胞进行了核型分析。自1976年起，圣地亚哥动物园开始保存来自极度濒危物种的生物标本，以便于将这些标本用作后代的研究材料;该项目被命名为“冷冻动物园”。这一项目的意义得到了国际认可，类似的项目，如英国的冷冻方舟项目，也在进行中。从极度濒危动物细胞培养的建立可能有助于这些冷冻保存项目，通过增加冷冻保存材料的稳定性和促进培养细胞的有效扩增。由于培养的细胞具有完整的极度濒危动物遗传信息，因此这些细胞有可能用于后代的遗传分析。橄榄里德利海龟在名古屋港公共水族馆饲养。从两只橄榄里德利海龟的鳍状鳍获得小的（3× 3 mm）皮肤组织活检标本。将组织活检标本立即浸入细胞培养基中。活检过程由名古屋港公共水族馆的兽医监督。对于原代培养，用I型胶原包被六孔细胞培养皿。我们之前的研究（Fukuda等人，2012）中描述了胶原蛋白涂层的详细方法。将细胞培养物保持在26 ℃、5%CO2下的加湿室中
The number of sea turtles in the wild is decreasing because of human activities, such as fishery bycatch (Peckham et al. 2007), oil spills, and marine pollution (Witherington 2001). Furthermore, illegal hunting for their oil, meat, and shells are still continuing (Koch et al. 2013). Hence, protection measures are being implemented in various countries to conserve the endangered species of sea turtles. In the past few decades, a major concern regarding sea turtles in the wild has been hybridization. Interspecific hybridization has been reported in several areas of the world (Karl et al. 1995; Barber et al. 2003; Lara-Ruiz et al. 2006). The original sea turtle species, such as hawksbill, loggerhead, and olive ridley, had established as independent species for more than millions of years despite overlaps among their habitats (Bowen et al. 1993). However, recent reports on intensive hybridization among these species clearly indicate that these unique original species are at a high risk of extinction in the near future. Acquiring information about the basic genetic background of these species, such as karyotype, is necessary to understand the cause of this intensive hybridization. In this study, we established a primary culture from olive ridley sea turtles and determined the karyotype of the primary cells. Since 1976, the San Diego Zoo started the preservation of biological specimens derived from critically endangered species in order to facilitate the use of these specimens as research materials for the following generations; this project was named “Frozen Zoo.” The significance of this project was internationally recognized, and similar projects, such as the Frozen Ark project in the UK, are underway. The establishment of cell cultures from critically endangered animals might contribute to these cryopreservation projects by increasing the stability of cryopreserved materials and facilitating efficient expansion of cultured cells. Since cultured cells have intact genetic information of the critically endangered animals, the cells have a potential to be used for the genetic analysis of future generations. The olive ridley sea turtles were maintained at the Port of Nagoya Public Aquarium. Small (3× 3 mm) dermal tissue biopsy specimens were obtained from the flipper-like fin of two olive ridley sea turtles. The tissue biopsy specimens were immediately immersed in the cell culture medium. The biopsy process was supervised by a veterinary doctor of the Port of Nagoya Public Aquarium. For the primary culture, a six-well cell culture dish was coated with type I collagen. The detailed method for collagen coating has been described in our previous study (Fukuda et al. 2012). The cell culture was maintained at 26 C under 5% CO2 in a humidified chamber