A Toolkit for Rapid Modular Construction of Biological Circuits in Mammalian Cells

Mapping Intimacies ◽

10.1101/506188 ◽

2018 ◽

Author(s):

João Pedro Fonseca ◽

Alain R. Bonny ◽

G. Renuka Kumar ◽

Andrew H. Ng ◽

Jason Town ◽

...

Keyword(s):

Mammalian Cells ◽

Ebola Virus ◽

Transcriptional Unit ◽

Biological Research ◽

Modular Construction ◽

Complex Circuits ◽

Dna Vectors ◽

Cellular Circuits ◽

Biological Circuits ◽

Different Characteristics

AbstractThe ability to rapidly assemble and prototype cellular circuits is vital for biological research and its applications in biotechnology and medicine. Current methods that permit the assembly of DNA circuits in mammalian cells are laborious, slow, expensive and mostly not permissive of rapid prototyping of constructs. Here we present the Mammalian ToolKit (MTK), a Golden Gate-based cloning toolkit for fast, reproducible and versatile assembly of large DNA vectors and their implementation in mammalian models. The MTK consists of a curated library of characterized, modular parts that can be easily mixed and matched to combinatorially assemble one transcriptional unit with different characteristics, or a hierarchy of transcriptional units weaved into complex circuits. MTK renders many cell engineering operations facile, as showcased by our ability to use the toolkit to generate single-integration landing pads, to create and deliver libraries of protein variants and sgRNAs, and to iterate through Cas9-based prototype circuits. As a biological proof of concept, we used the MTK to successfully design and rapidly construct in mammalian cells a challenging multicistronic circuit encoding the Ebola virus (EBOV) replication complex. This construct provides a non-infectious biosafety level 2 (BSL2) cellular assay for exploring the transcription and replication steps of the EBOV viral life cycle in its host. Its construction also demonstrates how the MTK can enable important and time sensitive applications such as the rapid testing of pharmacological inhibitors of emerging BSL4 viruses that pose a major threat to human health.

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A Toolkit for Rapid Modular Construction of Biological Circuits in Mammalian Cells

ACS Synthetic Biology ◽

10.1021/acssynbio.9b00322 ◽

2019 ◽

Vol 8 (11) ◽

pp. 2593-2606 ◽

Cited By ~ 4

Author(s):

João Pedro Fonseca ◽

Alain R. Bonny ◽

G. Renuka Kumar ◽

Andrew H. Ng ◽

Jason Town ◽

...

Keyword(s):

Mammalian Cells ◽

Modular Construction ◽

Biological Circuits

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Gene replacement with one-sided homologous recombination

Molecular and Cellular Biology ◽

10.1128/mcb.12.1.360-367.1992 ◽

1992 ◽

Vol 12 (1) ◽

pp. 360-367

Author(s):

N Berinstein ◽

N Pennell ◽

C A Ottaway ◽

M J Shulman

Keyword(s):

Homologous Recombination ◽

Dna Sequences ◽

Mammalian Cells ◽

Target Gene ◽

Gene Replacement ◽

Immunoglobulin Genes ◽

Nonhomologous Recombination ◽

Immunoglobulin Locus ◽

Chromosomal Sequences ◽

Dna Vectors

Homologous recombination is now routinely used in mammalian cells to replace endogenous chromosomal sequences with transferred DNA. Vectors for this purpose are traditionally constructed so that the replacement segment is flanked on both sides by DNA sequences which are identical to sequences in the chromosomal target gene. To test the importance of bilateral regions of homology, we measured recombination between transferred and chromosomal immunoglobulin genes when the transferred segment was homologous to the chromosomal gene only on the 3' side. In each of the four recombinants analyzed, the 5' junction was unique, suggesting that it was formed by nonhomologous, i.e., random or illegitimate, recombination. In two of the recombinants, the 3' junction was apparently formed by homologous recombination, while in the other two recombinants, the 3' junction as well as the 5' junction might have involved a nonhomologous crossover. As reported previously, we found that the frequency of gene targeting increases monotonically with the length of the region of homology. Our results also indicate that targeting with fragments bearing one-sided homology can be as efficient as with fragments with bilateral homology, provided that the overall length of homology is comparable. The frequency of these events suggests that the immunoglobulin locus is particularly susceptible to nonhomologous recombination. Vectors designed for one-sided homologous recombination might be advantageous for some applications in genetic engineering.

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Conserved Motifs within Ebola and Marburg Virus VP40 Proteins Are Important for Stability, Localization, and Subsequent Budding of Virus-Like Particles

Journal of Virology ◽

10.1128/jvi.02034-09 ◽

2009 ◽

Vol 84 (5) ◽

pp. 2294-2303 ◽

Cited By ~ 38

Author(s):

Yuliang Liu ◽

Luis Cocka ◽

Atsushi Okumura ◽

Yong-An Zhang ◽

J. Oriol Sunyer ◽

...

Keyword(s):

Self Assembly ◽

Mammalian Cells ◽

Ebola Virus ◽

Intracellular Localization ◽

Gel Filtration ◽

Point Mutations ◽

Marburg Virus ◽

Structure Stability ◽

Wild Type ◽

Virus Like Particles

ABSTRACT The filovirus VP40 protein is capable of budding from mammalian cells in the form of virus-like particles (VLPs) that are morphologically indistinguishable from infectious virions. Ebola virus VP40 (eVP40) contains well-characterized overlapping L domains, which play a key role in mediating efficient virus egress. L domains represent only one component required for efficient budding and, therefore, there is a need to identify and characterize additional domains important for VP40 function. We demonstrate here that the 96LPLGVA101 sequence of eVP40 and the corresponding 84LPLGIM89 sequence of Marburg virus VP40 (mVP40) are critical for efficient release of VP40 VLPs. Indeed, deletion of these motifs essentially abolished the ability of eVP40 and mVP40 to bud as VLPs. To address the mechanism by which the 96LPLGVA101 motif of eVP40 contributes to egress, a series of point mutations were introduced into this motif. These mutants were then compared to the eVP40 wild type in a VLP budding assay to assess budding competency. Confocal microscopy and gel filtration analyses were performed to assess their pattern of intracellular localization and ability to oligomerize, respectively. Our results show that mutations disrupting the 96LPLGVA101 motif resulted in both altered patterns of intracellular localization and self-assembly compared to wild-type controls. Interestingly, coexpression of either Ebola virus GP-WT or mVP40-WT with eVP40-ΔLPLGVA failed to rescue the budding defective eVP40-ΔLPLGVA mutant into VLPs; however, coexpression of eVP40-WT with mVP40-ΔLPLGIM successfully rescued budding of mVP40-ΔLPLGIM into VLPs at mVP40-WT levels. In sum, our findings implicate the LPLGVA and LPLGIM motifs of eVP40 and mVP40, respectively, as being important for VP40 structure/stability and budding.

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Morphology and Phenotype of Peripheral Erythrocytes of Fish: A Rapid Screening of Images by Using Software

International Letters of Natural Sciences ◽

10.18052/www.scipress.com/ilns.54.27 ◽

2016 ◽

Vol 54 ◽

pp. 27-41

Author(s):

Soumendra Nath Talapatra ◽

Priyadarshini Mitra ◽

Snehasikta Swarnakar

Keyword(s):

Image Analysis ◽

Mammalian Cells ◽

Yeast Cells ◽

Rapid Screening ◽

Biological Study ◽

Biological Research ◽

Analysis Software ◽

Image Analysis Software ◽

Comparative Results ◽

Cellular Phenotypes

Many information of biological study as stained cells analysis under microscope cannot be obtained rich information like detail morphology, shape, size, proper intensity etc. but image analysis software can easily be detected all these parameters within short duration. The cells types can be yeast cells to mammalian cells. An attempt has been made to detect cellular abnormalities from an image of metronidazole (MTZ) treated compared to control images of peripheral erythrocytes of fish by using non-commercial, open-source, CellProfiler (CP) image analysis software (Ver. 2.1.0). The comparative results were obtained after analysis the software. In conclusion, this image based screening of Giemsa stained fish erythrocytes can be a suitable tool in biological research for primary toxicity prediction at DNA level alongwith cellular phenotypes. Moreover, still suggestions are needed in relation to accuracy of present analysis for Giemsa stained fish erythrocytes because previous works have been carried out images of cells with fluorescence dye.

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New insights of CRISPR technology in human pathogenic fungi

Future Microbiology ◽

10.2217/fmb-2019-0183 ◽

2019 ◽

Vol 14 (14) ◽

pp. 1243-1255 ◽

Cited By ~ 1

Author(s):

Elvira Román ◽

Daniel Prieto ◽

Rebeca Alonso-Monge ◽

Jesús Pla

Keyword(s):

Mammalian Cells ◽

Genetic Manipulation ◽

Pathogenic Fungi ◽

Biological Research ◽

Therapeutic Approaches ◽

New Therapeutic Approaches ◽

Short Palindromic Repeat ◽

Future Outcomes ◽

Near Future ◽

Genome Manipulation

Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas systems have emerged as a powerful tool for genome manipulation. Class 2 type II CRISPR/ CAS9 is so far the most studied system and has been implemented in many biological systems such as mammalian cells, plants, fungi and bacteria. Fungi are important causes of human diseases worldwide. Genetic manipulation of pathogenic fungi is critical to develop new therapeutic approaches and novel antifungals. We will review here the progress done with CRISPR/ CAS9 systems in human pathogenic fungi, with emphasis in Candida albicans and the main modifications that have improved their usefulness in biological research. We finally discuss possible future outcomes and applications to the developed in a near future.

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Empirical Factors Affecting the Quality of Non-Negative Matrix Factorization of Mammalian Cell Raman Spectra

Applied Spectroscopy ◽

10.1177/0003702817732117 ◽

2017 ◽

Vol 71 (12) ◽

pp. 2681-2691 ◽

Cited By ~ 3

Author(s):

H. Georg Schulze ◽

Stanislav O. Konorov ◽

James M. Piret ◽

Michael W. Blades ◽

Robin F. B. Turner

Keyword(s):

Raman Spectra ◽

Mammalian Cell ◽

Matrix Factorization ◽

Mammalian Cells ◽

Hyperspectral Data ◽

Spectral Unmixing ◽

Biological Research ◽

Data Set ◽

Non Negative Matrix Factorization

Mammalian cells contain various macromolecules that can be investigated non-invasively with Raman spectroscopy. The particular mixture of major macromolecules present in a cell being probed are reflected in the measured Raman spectra. Determining macromolecular identities and estimating their concentrations from these mixture Raman spectra can distinguish cell types and otherwise enable biological research. However, the application of canonical multivariate methods, such as principal component analysis (PCA), to perform spectral unmixing yields mathematical solutions that can be difficult to interpret. Non-negative matrix factorization (NNMF) improves the interpretability of unmixed macromolecular components, but can be difficult to apply because ambiguities produced by overlapping Raman bands permit multiple solutions. Furthermore, theoretically sound methods can be difficult to implement in practice. Here we examined the effects of a number of empirical approaches on the quality of NNMF results. These approaches were evaluated on simulated mammalian cell Raman hyperspectra and the results were used to develop an enhanced procedure for implementing NNMF. We demonstrated the utility of this procedure using a Raman hyperspectral data set measured from human islet cells to recover the spectra of insulin and glucagon. This was compared to the relatively inferior PCA of these data.

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Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of Ebola Virus

Journal of Virology ◽

10.1128/jvi.01087-15 ◽

2015 ◽

Vol 89 (18) ◽

pp. 9440-9453 ◽

Cited By ~ 47

Author(s):

Emmanuel Adu-Gyamfi ◽

Kristen A. Johnson ◽

Mark E. Fraser ◽

Jordan L. Scott ◽

Smita P. Soni ◽

...

Keyword(s):

Plasma Membrane ◽

Host Cell ◽

Human Cell ◽

Mammalian Cells ◽

Matrix Protein ◽

Ebola Virus ◽

Cell Plasma Membrane ◽

Replication Process ◽

Cell Plasma

ABSTRACTLipid-enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. Ebola virus, which buds from the plasma membrane of the host cell, causes viral hemorrhagic fever and has a high fatality rate. To date, little has been known about how budding and egress of Ebola virus are mediated at the plasma membrane. We have found that the lipid phosphatidylserine (PS) regulates the assembly of Ebola virus matrix protein VP40. VP40 binds PS-containing membranes with nanomolar affinity, and binding of PS regulates VP40 localization and oligomerization on the plasma membrane inner leaflet. Further, alteration of PS levels in mammalian cells inhibits assembly and egress of VP40. Notably, interactions of VP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress, whereas PS is typically found only on the inner leaflet. Taking the data together, we present a model accounting for the role of plasma membrane PS in assembly of Ebola virus-like particles.IMPORTANCEThe lipid-enveloped Ebola virus causes severe infection with a high mortality rate and currently lacks FDA-approved therapeutics or vaccines. Ebola virus harbors just seven genes in its genome, and there is a critical requirement for acquisition of its lipid envelope from the plasma membrane of the human cell that it infects during the replication process. There is, however, a dearth of information available on the required contents of this envelope for egress and subsequent attachment and entry. Here we demonstrate that plasma membrane phosphatidylserine is critical for Ebola virus budding from the host cell plasma membrane. This report, to our knowledge, is the first to highlight the role of lipids in human cell membranes in the Ebola virus replication cycle and draws a clear link between selective binding and transport of a lipid across the membrane of the human cell and use of that lipid for subsequent viral entry.

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Expression Profiling of Single Mammalian Cells – Small is Beautiful

Yeast ◽

10.1002/1097-0061(20000930)17:3<211::aid-yea26>3.0.co;2-7 ◽

2000 ◽

Vol 1 (3) ◽

pp. 211-217 ◽

Cited By ~ 31

Author(s):

Gerard Brady

Keyword(s):

Mrna Expression ◽

Single Cell ◽

Mammalian Cells ◽

Single Cell Analysis ◽

Single Cells ◽

Expression Patterns ◽

Global Gene Expression ◽

Small Samples ◽

Biological Research ◽

Cell Expression

Increasingly mRNA expression patterns established using a variety of molecular technologies such as cDNA microarrays, SAGE and cDNA display are being used to identify potential regulatory genes and as a means of providing valuable insights into the biological status of the starting sample. Until recently, the application of these techniques has been limited to mRNA isolated from millions or, at very best, several thousand cells thereby restricting the study of small samples and complex tissues. To overcome this limitation a variety of amplification approaches have been developed which are capable of broadly evaluating mRNA expression patterns in single cells. This review will describe approaches that have been employed to examine global gene expression patterns either in small numbers of cells or, wherever possible, in actual isolated single cells. The first half of the review will summarize the technical aspects of methods developed for single-cell analysis and the latter half of the review will describe the areas of biological research that have benefited from single-cell expression analysis.

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Mitochondrial form, function and signalling in aging

Biochemical Journal ◽

10.1042/bcj20160451 ◽

2016 ◽

Vol 473 (20) ◽

pp. 3421-3449 ◽

Cited By ~ 18

Author(s):

Ignacio Amigo ◽

Fernanda M. da Cunha ◽

Maria Fernanda Forni ◽

Wilson Garcia-Neto ◽

Pâmela A. Kakimoto ◽

...

Keyword(s):

Mammalian Cells ◽

Energy Homeostasis ◽

Cell Types ◽

Redox Balance ◽

Whole Body ◽

Model Organisms ◽

Resistance To Stress ◽

Whole Body Metabolism ◽

And Function ◽

Different Characteristics

Aging is often accompanied by a decline in mitochondrial mass and function in different tissues. Additionally, cell resistance to stress is frequently found to be prevented by higher mitochondrial respiratory capacity. These correlations strongly suggest mitochondria are key players in aging and senescence, acting by regulating energy homeostasis, redox balance and signalling pathways central in these processes. However, mitochondria display a wide array of functions and signalling properties, and the roles of these different characteristics are still widely unexplored. Furthermore, differences in mitochondrial properties and responses between tissues and cell types, and how these affect whole body metabolism are also still poorly understood. This review uncovers aspects of mitochondrial biology that have an impact upon aging in model organisms and selected mammalian cells and tissues.

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