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expression of a gene from a BAC usually recapitulates splicing patterns and expression levels, including cellcycle controls. Because BACs often contain all cis regulatory elements for gene expression, and are usually integrated at low transgene copy number, the artifacts associated with dysregulated cDNA overexpression from strong (usually viral) promoters are far less likely to be encountered. The simplicity of the Kittler et al. strategy relies on two aspects. First, to facilitate selection of cells with BAC transgenes, Kittler et al. use recombineering [9,10] to put a selectable gene onto the BAC. Because the selectable gene carries a dual Escherichia coli/mammalian cell promoter, the generation of a selectable transgenic construct takes only one recombineering step [11]. This simple application of recombineering greatly enhances the utility of BACs as reagents in functional genomics. For the above reasons, it is likely that BACs will become preferred to cDNA expression vectors. Second, the rescuing transgenic mRNA must obviously differ from the mRNA targeted by RNAi. Therefore, Kittler et al. select BACs from a different species, in their case from the mouse. The great convenience of crossspecies rescue comes with a risk. At a frequency that will probably be very low, the cross-species gene/BAC will not rescue. Notably, the failure of cross-species BAC rescue will cast doubt on the RNAi specicity, rather than provoking a misleading functional conclusion. In these cases, recombineering-mediated point mutagenesis could be used to alter the RNAi target region in a BAC from the same species [12]. Although this is straightforward, it entails more work than simply choosing a cross-species BAC. Further implications Although it is a remarkable tool, RNAi can achieve only a loss of function. For example, it cannot achieve point mutagenesis or expression of fusion proteins. In a simple variation of the cross-species BAC rescue strategy, Kittler

et al. outline a way to achieve these goals using the combination of RNAi and BAC rescue. Rather than recombineering a BAC to carry a selectable gene, they recombineer the BAC to make a green ourescence protein (GFP) fusion with the rescuing protein. Thereby, the endogenous protein, knocked-down by RNAi, is not merely replaced by its cross-species counterpart but by a GFP fusion protein. Further variations of the same theme are obvious. The paper by Kittler et al. therefore not only describes the best workable control for the specicity of RNAi knock-downs so far, but also suggests a new general strategy for reverse genetic approaches, which will be applicable to many experimental systems.
References
1 Meister, G. and Tuschl, T. (2004) Mechanisms of gene silencing by double-stranded RNA. Nature 431, 343349 2 Carpenter, A.E. and Sabatini, D.M. (2004) Systematic genome-wide screens of gene function. Nat. Rev. Genet. 5, 1122 3 Hannon, G.J. and Rossi, J.J. (2004) Unlocking the potential of the human genome with RNA interference. Nature 431, 371378 4 Bridge, A.J. et al. (2003) Induction of an interferon response by RNAi vectors in mammalian cells. Nat. Genet. 34, 263264 5 Persengiev, S.P. et al. (2004) Nonspecic, concentration-dependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs). RNA 10, 1218 6 Elbashir, S.M. et al. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494498 7 Editorial. (2003) Whither RNAi? Nat. Cell. Biol. 5, 489490 8 Kittler, R. et al. (2005) RNA interference rescue by bacterial articial chromosome transgenesis in mammalian tissue culture cells. Proc. Natl. Acad. Sci. U. S. A. 102, 23962401 9 Zhang, Y. et al. (1998) A new logic for DNA engineering using recombination in E. coli. Nat. Genet. 20, 123128 10 Copeland, N.G. et al. (2001) Recombineering: a powerful new tool for mouse functional genomics. Nat. Rev. Genet. 2, 769779 11 Angrand, P-O. et al. (1999) Simplied generation of targeting constructs using ET recombination. Nucleic Acids Res. 27, e16 12 Yang, Y. and Sharan, S.K. (2003) A simple two-step, hit and x method to generate subtle mutations in BACs using short denatured PCR fragments. Nucleic Acids Res. 31, e80
0167-7799/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2005.06.007

Letters

Microalgae: the self-synchronized eukaryotes

Ana Otero1 and Ken Goto2
1 2

Departamento de Microbiologa y Parasitologa, Universidad de Santiago de Compostela, Santiago, E-15782, Spain Laboratory of Biological Rhythms, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan 080-8555

Owing to the increase of sensitivity of high-throughput techniques in proteomics and genomics, truly synchronized cultures should be a prerequisite for a reliable identication of key proteins and genes involved in the cell-division cycle (CDC), both in eukaryotes and prokaryotes. Recently, an interesting controversy regarding the accuracy and reliability of synchronization methods using
Corresponding author: Otero, A. (mpaotero@usc.es). Available online 13 June 2005
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whole-culture versus Helmstetters baby machine has been raised in this journal [1,2]. This letter does not discuss the benets and drawbacks of both methods but proposes a new tool to the scientic community requiring truly synchronized eukaryotic cells for CDC studies: the self-synchronized cultures of microalgae. Microalgae are photosynthetic eukaryotic microorganisms that exhibit a naturally phased cell division, which occurs only during a particular time of the day, generally

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at night [3,4]. Under optimal culture conditions, a complete synchronicity of cell division can be obtained with all cells dividing at the same time and the factorial increase of cell number being exactly 2.0. Daily lightdark (LD) cycles have been utilized to induce division synchrony in various algae including diatoms, dinoagellates and the model organisms Euglena gracilis and Chlamydomonas reinhardtii [3,4]. The maintenance of optimal culture conditions, especially light intensity and duration of the light period, rather than a selection of a homogeneous initial population, is the key factor. In most cases, a simple daily renewal of 50% of the culture to keep the optimal conditions is enough to maintain division synchrony indenitely [5]. Renewal of the culture media should be carried out at the end of the dark phase or very early during the light period to avoid perturbations in the synchronicity pattern [5,6]. In several green algae such as Chlorella, Scenedesmus or Chlamydomonas, each synchronous cell cycle might produce 2, 4, 8 or even 16 daughter cells by multiple ssion, depending on the species and/or culture conditions [3]. The use of synchronous cultures of microalgae presenting multiple ssion might cause difculties in the interpretation of results, as in the case of the absence of a neat nuclei division pattern observed by ow cytometry in Chlamydomonas [6]. Efforts would be warranted to achieve a factorial increase of exactly 2.0 in the green algae. The use of microalgae as model organisms for the study of CDC still offers another interesting feature: the close interaction between cell division and circadian rhythms [4,7]. Since the nding of the circadian phasing of the cell division in the dinoagellate Lingulodinium polyedrum (formerly Gonyaulax polyedra) under a constant condition without an external time-cue by Sweeney and Hastings in 1958 (see Ref. [4]), it has been documented in various organisms from cyanobacteria to humans. We now know in E. gracilis that a circadian clock prevents developmentally matured G2-phase cells from progressing to M phase between 4 h before the onset and the end of a subjective or endogenous day [8]. A circadian rhythm responds to, and runs in phase with 24-h LD cycles such that a subjective night comes exactly during the dark period, not excepting the timing of cell division; thus, light only serves as a timecue that daily resets the circadian rhythms [9]. Algal growth, most often the G1 progression, requires photosynthetic light energy. Nevertheless, at least in E. gracilis [10], the G2-to-M transition might also involve photosynthesis: darkness arrests the transition, unless G2 cells are sufciently irradiated beforehand to acquire the capability of undergoing the transition in the subsequent darkness; photosynthesis is required here for a signal, but not a metabolic demand. Importantly, a circadian rhythm regulates the light responses, such that irradiation around a subjective dusk maximally induces the capability of the

G2-toM transition in the dark, whereas one around a subjective dawn does not. The nding predicts that a good synchrony might require the photoperiod to be close to, or more than, 12 h. It is to be noted that the synchronization of microalgal CDC through LD cycles involves the interaction of three components: a direct response to light or darkness, cellcycle dependent phenomena and circadian rhythms. In photoautotrophic cultures, a phenomenon either intrinsic to CDC or related to its direct effect disappears when the alga is placed into continuous darkness, whereas circadian rhythms still persist. Thus, we can extract a phenomenon truly dependent on either CDC or circadian clocks, offering great opportunities both to those studying CDC and to those studying circadian rhythms. Microalgae are a source of numerous compounds of biotechnological interest and their productivity often depends on the cell-cycle phase and/or circadian phase. In addition, the recent advances in algal genomics and the incipient development of methods for the stable genetic manipulation of microalgae [11,12], together with unique features such as the presence of strong circadian rhythms that are maintained even in outer space, have increased interest in the use of these fascinating microorganisms as models.
References
1 Cooper, S. (2004) Is whole-culture synchronization biologys perpetual motion machine? Trends Biotechnol. 22, 266269 2 Spellman, P.T. and Sherlock, G. (2004) Reply: whole-culture synchronization effective tools for cell cycle studies. Trends Biotechnol. 22, 270273 3 Tamiya, H. (1966) Synchronous cultures of algae. Annu. Rev. Plant Physiol. 17, 126 4 Edmunds, L.N., Jr, ed. (1988) Cellular and Molecular Bases of Biological Clocks, Springer-Verlag 5 Fabregas, J. et al. (1995) Productivity and biochemical composition of cyclostat cultures of the marine microalga Tetraselmis suecica. Appl. Microbiol. Biotechnol. 43, 617621 6 Lemaire, S.D. et al. (1999) Analysis of light/dark synchronization of cell-wall-less Chlamydomonas reinhardtii (Chlorophyta) cells by ow cytometry. Eur. J. Phycol. 34, 279286 7 Mittag, M. (2001) Circadian rhythms in microalgae. Int. Rev. Cytol. 206, 213247 8 Bolige, A. et al. (2005) Circadian G2-arrest as related to circadian gating of cell population growth in Euglena. Plant Cell Physiol. 46, 931936 9 Johnson, C.H. et al. (2003) Entrainment of circadian programs. Chronobiol. Int. 20, 741774 10 Hagiwara, S. et al. (2002) Circadian gating of photoinduction of commitment to cell-cycle transitions in relation to photoperiodic control of cell reproduction in Euglena. Photochem. Photobiol. 76, 105115 11 Leon-Banares, R. et al. (2004) Transgenic microalgae as green cellfactories. Trends Biotechnol. 22, 4552 12 Grossman, A.R. (2005) Paths toward algal genomics. Plant Physiol. 137, 410427
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