The first evidence for genotypic stability in a cryopreserved transgenic diatom
The first evidence for genotypic stability in a cryopreserved transgenic diatom
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DOI：
10.1007/s10811-013-0047-y
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发表时间：
2014-02
影响因子：
3.3
通讯作者：
Rachel Hipkin;J. Day;C. Rad-Menéndez;T. Mock
中科院分区：
文献类型：
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作者：
Rachel Hipkin;J. Day;C. Rad-Menéndez;T. Mock
未来的藻类生物技术将需要增强的生产菌株，能够更快地生长，更有效地转换太阳能和/或更高水平的代谢产物生产。几乎可以肯定的是，转基因生物将被用于确保成本效益高、经济上可行的一系列代谢产物的生产。与所有生物技术过程一样，生产菌株的功能稳定性、可靠性和安全性在藻类生物技术中至关重要，因为没有这些，任何生物技术过程都是不可持续的。本研究采用传统的、低技术含量的、依数性的低温保存方法对转基因模式菌株海链藻CCAP 1085/23进行了低温保存。这采用二甲基亚砜[5%（v/v）]作为冷冻保护剂，使用两步冷却方法：初始控制速率冷却，然后浸入液氮中。获得了高水平的解冻后活力（70- 85%），并且在回收冷冻保存的材料时，没有观察到插入基因（big 1-GFP）的蛋白质的表达降低。此外，冷冻保存不影响BIG 1-GFP蛋白的定位，如染色样品的显微镜所示，也不影响其功能，如Western印迹所示。
Future algal biotechnology will need enhanced production strains, capable of more rapid growth, more efficient solar-energy conversion and/or higher levels of metabolite production. Almost certainly transgenic organisms will be used to ensure the cost-effective, economically viable production of a range of metabolites. As with all biotechnological processes, the functional stability, reliability and security of the production strains will be of paramount importance in algal biotechnology, as without this no biotechnological process is sustainable. In this study, the transgenic model strainThalassiosira pseudonanaCCAP 1085/23 was cryopreserved using a conventional, low-tech, colligative cryopreservation protocol. This employed dimethyl sulphoxide [5 % (v/v)] as a cryoprotectant, using a two-step cooling approach: initial controlled-rate cooling, followed by plunging into liquid nitrogen. High levels of post-thaw viability (70–85 %) were obtained, and on recovery of cryopreserved material no reduction in expression of the protein of the inserted gene (big1-GFP) was observed. Additionally, cryopreservation does not affect the localisation of the BIG1-GFP protein as demonstrated by microscopy of stained samples, nor its functionality as demonstrated by Western blotting.