The first evidence for genotypic stability in a cryopreserved transgenic diatom
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 strain Thalassiosira pseudonana CCAP 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.
KeywordsAlgal biotechnology Cryopreservation Genotypic stability Passive freezer Transgenic diatom
Rachel Hipkin was supported by a Norwich Research Park PhD studentship. John Day and Cecilia Rad-Menéndez acknowledge National Capability funding for the CCAP from NERC. Thomas Mock acknowledges funding from NERC (NE/J013730/1), the University of East Anglia for a proof-of-principle grant and Nicole Poulsen for providing vectors and advice regarding nuclear transformation of Thalassiosira pseudonana.
- Armbrust EV, Berges JA, Bowler C, Green BR, Martinez D, Putnam NH, Zhou S, Allen AE, Apt KE, Bechner M, Brzezinski MA, Chaal BK, Chiovitti A, Davis AK, Demarest MS, Detter JC, Glavina T, Goodstein D, Hadi MZ, Hellsten U, Hildebrand M, Jenkins BD, Jurka J, Kapitonov VV, Kröger N, Lau WWY, Lane TW, Larimer FW, Lippmeier JC, Lucas S, Medina M, Montsant A, Obornik M, Parker MS, Palenik B, Pazour GJ, Richardson PM, Rynearson TA, Valentin K, Vardi A, Wilkerson FP, Rokhsar DS (2004) The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism. Science 306:79–86PubMedCrossRefGoogle Scholar
- Day JG (2004) Cryopreservation: fundamentals, mechanisms of damage on freeze/thawing and application in culture collections. Nova Hedwigia 79:191–205Google Scholar
- Day JG, Brand JJ (2005) Cryopreservation methods for maintaining cultures. In: Andersen RA (ed) Algal culturing techniques. Academic, New York, pp 165–187Google Scholar
- Day JG, Pröschold T, Friedl T, Lorenz M, Silva PC (2010) Conservation of microalgal type material: approaches needed for 21st century science. Taxon 59:3–6Google Scholar
- Harrison PJ, Waters RE, Taylor FJR (1980) A broad spectrum artificial medium for coastal and open ocean phytoplankton. J Phycol 16:28–35Google Scholar
- Lorenz M, Friedl T, Day JG (2005) Perpetual maintenance of actively metabolizing microalgal cultures. In: Andersen RA (ed) Algal culturing techniques. Academic, New York, pp 145–155Google Scholar
- Pearson BM, Jackman PJH, Painting KA, Morris GJ (1990) Stability of genetically manipulated yeasts under different cryopreservation regimes. CryoLetters 11:205–210Google Scholar
- Rad-Menéndez C (2011) Phenotypic and genotypic characterization of Thalassiosira pseudonana (Bacillariophyta) strains. MSc thesis, University of Aberdeen, 133pp.Google Scholar