A fluorescence-activated cell sorting-based strategy for rapid isolation of high-lipid Chlamydomonas mutants.
A fluorescence-activated cell sorting-based strategy for rapid isolation of high-lipid Chlamydomonas mutants.
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DOI：
10.1111/tpj.12682
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发表时间：
2015-01
期刊：
影响因子：
--
通讯作者：
Jonikas MC
中科院分区：
文献类型：
--
作者：
Terashima M;Freeman ES;Jinkerson RE;Jonikas MC
人们对养殖藻类直接生产生物燃料和有价值的脂类很感兴趣。莱茵衣藻（Chlamydomonas reinhardtii）是研究绿藻脂质代谢的主要模型系统，但目前分离这种脂质含量受到干扰的生物突变体的方法缓慢而繁琐。在这里，我们提出了衣藻高脂分选（CHiLiS）策略，该策略可以通过荧光激活细胞分选（FACS）富集高脂突变体，这些突变体用脂敏感染料尼罗红染色。该方法从诱变到分离突变体仅需5周。我们开发了一种染色方案，允许定量脂质含量，同时保持细胞活力。我们利用每个细胞的叶绿素荧光作为内部控制，改进了高脂突变体与野生型的分离。我们最初证明，从sta1和野生型细胞的混合物中，已知的高脂突变体sta1富集了20倍。然后，我们应用CHiLiS从大约6万个突变体中对数千个高脂细胞进行分类。用这种方法分离的24个突变体的流式细胞术分析显示，约50%的突变体表现出可重复的高脂表型。我们进一步通过火焰电离检测和质谱脂质组学鉴定了9个脂质含量最高的突变体。分析的所有突变体均具有较高的三酰甘油含量和全细胞脂肪酸组成紊乱。一个任意选择的突变体在显微镜下被评估，显示比野生型更大的脂滴。前所未有的CHiLiS吞吐量打开了大门，以系统级的理解绿藻脂质生物学，使基因组饱和分离突变的关键基因。
There is significant interest in farming algae for the direct production of biofuels and valuable lipids. Chlamydomonas reinhardtii is the leading model system for studying lipid metabolism in green algae, but current methods for isolating mutants of this organism with a perturbed lipid content are slow and tedious. Here, we present the Chlamydomonas high-lipid sorting (CHiLiS) strategy, which enables enrichment of high-lipid mutants by fluorescence-activated cell sorting (FACS) of pooled mutants stained with the lipid-sensitive dye Nile Red. This method only takes 5 weeks from mutagenesis to mutant isolation. We developed a staining protocol that allows quantification of lipid content while preserving cell viability. We improved separation of high-lipid mutants from the wild type by using each cell's chlorophyll fluorescence as an internal control. We initially demonstrated 20-fold enrichment of the known high-lipid mutant sta1 from a mixture of sta1 and wild-type cells. We then applied CHiLiS to sort thousands of high-lipid cells from a pool of about 60 000 mutants. Flow cytometry analysis of 24 individual mutants isolated by this approach revealed that about 50% showed a reproducible high-lipid phenotype. We further characterized nine of the mutants with the highest lipid content by flame ionization detection and mass spectrometry lipidomics. All mutants analyzed had a higher triacylglycerol content and perturbed whole-cell fatty acid composition. One arbitrarily chosen mutant was evaluated by microscopy, revealing larger lipid droplets than the wild type. The unprecedented throughput of CHiLiS opens the door to a systems-level understanding of green algal lipid biology by enabling genome-saturating isolation of mutants in key genes.
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