A recombineering pipeline to clone large and complex genes in Chlamydomonas

Abstract 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 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 kilobases. 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.

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A recombineering pipeline to clone large and complex genes in Chlamydomonas

10.1101/2020.05.06.080416 ◽

2020 ◽

Author(s):

Tom Emrich-Mills ◽

Gary Yates ◽

James Barrett ◽

Irina Grouneva ◽

Chun Sing Lau ◽

...

Keyword(s):

Success Rate ◽

Fluorescent Protein ◽

Gc Content ◽

Research Community ◽

Mass Spectrometry Data ◽

Mutant Library ◽

Parallel Cloning ◽

Mutant Complementation ◽

Protein Tagging ◽

Genomic Gc Content

AbstractThe ability to clone genes has driven fundamental advances in cell and molecular biology, enabling researchers to introduce precise mutations, generate fluorescent protein fusions for localization and to confirm genetic causation by mutant complementation. Most gene cloning is PCR or DNA synthesis dependent, which can become costly and technically challenging as genes increase in size and particularly if they contain complex regions. This has been a long-standing challenge for the Chlamydomonas reinhardtii research community, with a high percentage of genes containing complex sequence structures, an average genomic GC content of 64% and gene expression requiring regular introns for stable transcription. Here we overcome these challenges via the development of a recombineering pipeline that enables the rapid parallel cloning of genes from a Chlamydomonas BAC collection. We show the method can successfully retrieve large and complex genes that PCR-based methods have previously failed to clone, including genes as large as 23 kilobases, thus making previously technically challenging genes to study now amenable to cloning. We initially applied the pipeline to 12 targets with a 92% cloning success rate. We then developed a high-throughput approach and targeted 191 genes relating to the Chlamydomonas CO2 concentrating mechanism (CCM) with an overall cloning success rate of 77% that is independent of gene size. Localization of a subset of CCM targets has confirmed previous mass spectrometry data and identified new pyrenoid components. To expand the functionality of our system, we developed a series of localization vectors that enable complementation of Chlamydomonas Library Project mutants and enable protein tagging with a range of fluorophores. Vectors and detailed protocols are available to facilitate the easy adoption of this method by the Chlamydomonas research community. We envision that this technology will open up new possibilities in algal and plant research and be complementary to the Chlamydomonas mutant library.

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Characterization of bacterial artificial chromosome transgenic mice expressing mCherry fluorescent protein substituted for the murine smooth muscle α-actin gene

genesis ◽

10.1002/dvg.20638 ◽

2010 ◽

Vol 48 (7) ◽

pp. 457-463 ◽

Cited By ~ 17

Author(s):

John J. Armstrong ◽

Irina V. Larina ◽

Mary E. Dickinson ◽

Warren E. Zimmer ◽

Karen K. Hirschi

Keyword(s):

Smooth Muscle ◽

Bacterial Artificial Chromosome ◽

Transgenic Mice ◽

Fluorescent Protein ◽

Artificial Chromosome ◽

Actin Gene

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Visualization of lymphatic vessels by Prox1-promoter directed GFP reporter in a bacterial artificial chromosome-based transgenic mouse

Blood ◽

10.1182/blood-2010-07-298562 ◽

2011 ◽

Vol 117 (1) ◽

pp. 362-365 ◽

Cited By ~ 128

Author(s):

Inho Choi ◽

Hee Kyoung Chung ◽

Swapnika Ramu ◽

Ha Neul Lee ◽

Kyu Eui Kim ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Bacterial Artificial Chromosome ◽

Transgenic Mouse ◽

Fluorescent Protein ◽

Lymphatic Vessels ◽

Artificial Chromosome ◽

Transgenic Animal ◽

Lymphatic Development ◽

Vasculogenesis And Angiogenesis ◽

Green Fluorescent

Abstract Although the blood vessel-specific fluorescent transgenic mouse has been an excellent tool to study vasculogenesis and angiogenesis, a lymphatic-specific fluorescent mouse model has not been established to date. Here we report a transgenic animal model that expresses the green fluorescent protein under the promoter of Prox1, a master control gene in lymphatic development. Generated using an approximately 200-kb-long bacterial artificial chromosome harboring the entire Prox1 gene, this Prox1-green fluorescent protein mouse was found to faithfully recapitulate the expression pattern of the Prox1 gene in lymphatic endothelial cells and other Prox1-expressing organs, and enabled us to conveniently visualize detailed structure and morphology of lymphatic vessels and networks throughout development. Our data demonstrate that this novel transgenic mouse can be extremely useful for detection, imaging, and isolation of lymphatic vessels and monitoring wound-associated lymphangiogenesis. Together, this Prox1-green fluorescent protein transgenic mouse will be a great tool for the lymphatic research.

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Shaping a Plant Genetics and Molecular Biology research community in Brazil

Genetics and Molecular Biology ◽

10.1590/1678-4685-gmb-2017-00050001 ◽

2017 ◽

Vol 40 (1 suppl 1) ◽

pp. I-I

Author(s):

Marie-Anne Van Sluys

Keyword(s):

Molecular Biology ◽

Research Community ◽

Plant Genetics ◽

Biology Research

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matchms - processing and similarity evaluation of mass spectrometry data

10.1101/2020.08.06.239244 ◽

2020 ◽

Cited By ~ 1

Author(s):

Florian Huber ◽

Stefan Verhoeven ◽

Christiaan Meijer ◽

Hanno Spreeuw ◽

Efraín Manuel Villanueva Castilla ◽

...

Keyword(s):

Mass Spectrometry ◽

Open Access ◽

High Throughput ◽

Life Sciences ◽

Research Community ◽

Significant Fraction ◽

Mass Spectrometry Data ◽

Joint Effort ◽

Complex Datasets

SummaryMass spectrometry data is at the heart of numerable applications in the biomedical and life sciences. With growing use of high throughput techniques researchers need to analyse larger and more complex datasets. In particular through joint effort in the research community, fragmentation mass spectrometry datasets are growing in size and number. Platforms such as MassBank (Horai et al. 2010), GNPS (Wang et al. 2016) or MetaboLights (Haug et al. 2020) serve as an open-access hub for sharing of raw, processed, or annotated fragmentation mass spectrometry data (MS/MS). Without suitable tools, however, exploitation of such datasets remains overly challenging. In particular, large collected datasets contain data aquired using different instruments and measurement conditions, and can further contain a significant fraction of inconsistent, wrongly labeled, or incorrect metadata (annotations).

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MassComp, a lossless compressor for mass spectrometry data

10.1101/542894 ◽

2019 ◽

Cited By ~ 1

Author(s):

Ruochen Yang ◽

Xi Chen ◽

Idoia Ochoa

Keyword(s):

Mass Spectrometry ◽

Numerical Data ◽

Mass Spectrometry Data ◽

Compression Algorithms ◽

Compression Performance ◽

Average Improvement ◽

The Family ◽

Efficient Representation ◽

Cost Efficient ◽

Biology Research

Background: Mass Spectrometry (MS) is a widely used technique in biology research, and has become key in proteomics and metabolomics analyses. As a result, the amount of MS data has significantly increased in recent years. For example, the MS repository MassIVE contains more than 123TB of data. Somehow surprisingly, these data are stored uncompressed, hence incurring a significant storage cost. Efficient representation of these data is therefore paramount to lessen the burden of storage and facilitate its dissemination. Results We present MassComp, a lossless compressor optimized for the numerical (m/z)-intensity pairs that account for most of the MS data. We tested MassComp on several MS data and show that it delivers on average a 46% reduction on the size of the numerical data, and up to 89%. These results correspond to an average improvement of more than 27% when compared to the general compressor gzip and of 40% when compared to the state-of-the-art numerical compressor FPC. When tested on entire files retrieved from the MassIVE repository, MassComp achieves on average a 59% size reduction. MassComp is written in C++ and freely available at https://github.com/iochoa/MassComp. Conclusions: The compression performance of MassComp demonstrates its potential to significantly reduce the footprint of MS data, and shows the benefits of designing specialized compression algorithms tailored to MS data. MassComp is an addition to the family of omics compression algorithms designed to lessen the storage burden and facilitate the exchange and dissemination of omics data.

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Cloning and Mutagenesis of the Murine Gammaherpesvirus 68 Genome as an Infectious Bacterial Artificial Chromosome

Journal of Virology ◽

10.1128/jvi.74.15.6964-6974.2000 ◽

2000 ◽

Vol 74 (15) ◽

pp. 6964-6974 ◽

Cited By ~ 267

Author(s):

Heiko Adler ◽

Martin Messerle ◽

Markus Wagner ◽

Ulrich H. Koszinowski

Keyword(s):

Bacterial Artificial Chromosome ◽

Fluorescent Protein ◽

Artificial Chromosome ◽

Mouse Strains ◽

Viral Gene ◽

Wild Type ◽

Murine Gammaherpesvirus ◽

Host Interaction ◽

Gammaherpesvirus 68 ◽

Murine Gammaherpesvirus 68

ABSTRACT Gammaherpesviruses cause important infections of humans, in particular in immunocompromised patients. Recently, murine gammaherpesvirus 68 (MHV-68) infection of mice has been developed as a small animal model of gammaherpesvirus pathogenesis. Efficient generation of mutants of MHV-68 would significantly contribute to the understanding of viral gene functions in virus-host interaction, thereby further enhancing the potential of this model. To this end, we cloned the MHV-68 genome as a bacterial artificial chromosome (BAC) inEscherichia coli. During propagation in E. coli, spontaneous recombination events within the internal and terminal repeats of the cloned MHV-68 genome, affecting the copy number of the repeats, were occasionally observed. The gene for the green fluorescent protein was incorporated into the cloned BAC for identification of infected cells. BAC vector sequences were flanked byloxP sites to allow the excision of these sequences using recombinase Cre and to allow the generation of recombinant viruses with wild-type genome properties. Infectious virus was reconstituted from the BAC-cloned MHV-68. Growth of the BAC-derived virus in cell culture was indistinguishable from that of wild-type MHV-68. To assess the feasibility of mutagenesis of the cloned MHV-68 genome, a mutant virus with a deletion of open reading frame 4 was generated. Genetically modified MHV-68 can now be analyzed in functionally modified mouse strains to assess the role of gammaherpesvirus genes in virus-host interaction and pathogenesis.

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