A recombineering pipeline to clone large and complex genes in Chlamydomonas.
A recombineering pipeline to clone large and complex genes in Chlamydomonas.
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DOI：
10.1093/plcell/koab024
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发表时间：
2021-05-31
期刊：
影响因子：
--
通讯作者：
Mackinder LCM
中科院分区：
文献类型：
--
作者：
Emrich-Mills TZ;Yates G;Barrett J;Girr P;Grouneva I;Lau CS;Walker CE;Kwok TK;Davey JW;Johnson MP;Mackinder LCM
克隆基因的能力极大地促进了细胞和分子生物学研究，使研究人员能够产生荧光蛋白融合进行定位，并通过突变互补来确认遗传原因。大多数基因克隆是聚合酶链式反应或�合成依赖的，随着基因大小的增加，特别是如果它们包含复杂的区域，这可能会变得昂贵和技术上的挑战。这对莱茵衣藻研究界来说是一个长期的挑战，因为这种藻类有很高比例的基因包含复杂的序列结构。在这里，我们克服了这些挑战，开发了一种重组工程管道，用于从衣藻细菌人工染色体收集中快速并行克隆基因。为了产生用于定位的荧光蛋白融合，我们在批量和高通量规模上应用该管道对203个与衣藻二氧化碳浓缩机制(CCM)相关的基因进行了克隆，总体克隆成功率为77%。克隆的成功与基因大小和复杂性无关，克隆的基因长达23kb。CCM靶标的一个子集的定位证实了先前的质谱学数据，确定了新的类蛋白核成分，并实现了突变体的互补。我们提供了载体和详细的协议，以方便这项技术的采用，我们预计这将在藻类和植物研究中开辟新的可能性。开发了一种高通量系统，用于高频克隆大而复杂的基因，并对莱茵衣藻中的一系列荧光团进行突变互补和蛋白质标记。
The ability to clone genes has greatly advanced cell and molecular biology research, enabling researchers to generate fluorescent protein fusions for localization and confirm genetic causation by mutant complementation. Most gene cloning is polymerase chain reaction (PCR)�or DNA synthesis-dependent, which can become costly and technically challenging as genes increase in size, particularly if they contain complex regions. This has been a long-standing challenge for the Chlamydomonas reinhardtii research community, as this alga has a high percentage of genes containing complex sequence structures. Here we overcame these challenges by developing a recombineering pipeline for the rapid parallel cloning of genes from a Chlamydomonas bacterial artificial chromosome collection. To generate fluorescent protein fusions for localization, we applied the pipeline at both batch and high-throughput scales to 203 genes related to the Chlamydomonas CO2 concentrating mechanism (CCM), with an overall cloning success rate of 77%. Cloning success was independent of gene size and complexity, with cloned genes as large as 23 kb. Localization of a subset of CCM targets confirmed previous mass spectrometry data, identified new pyrenoid components, and enabled complementation of mutants. We provide vectors and detailed protocols to facilitate easy adoption of this technology, which we envision will open up new possibilities in algal and plant research. A high-throughput system was developed to clone large, complex genes at high frequency and perform mutant complementation and protein tagging with a range of fluorophores in Chlamydomonas reinhardtii.
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影响因子：
11.6
作者：
Brueggeman, Andrew J.;Gangadharaiah, Dayananda S.;Ladunga, Istvan
通讯作者：
Ladunga, Istvan
影响因子：
16.6
作者：
通讯作者：
--
影响因子：
48
作者：
Bindels, Daphne S.;Haarbosch, Lindsay;Gadella, Theodorus W. J., Jr.
通讯作者：
Gadella, Theodorus W. J., Jr.
影响因子：
14.9
作者：
Benson, G
通讯作者：
Benson, G
DOI：
10.1111/tpj.13033
发表时间：
2015-11
期刊：
The Plant journal : for cell and molecular biology
影响因子：
--
作者：
Barahimipour R;Strenkert D;Neupert J;Schroda M;Merchant SS;Bock R
通讯作者：
Bock R