scholarly journals Development of a robust transient expression screening system in protoplasts of Cannabis

2021 ◽  
Author(s):  
L. Matchett-Oates ◽  
Ehab Mohamaden ◽  
G. C. Spangenberg ◽  
N. O. I. Cogan
Keyword(s):  
Transient Expression ◽  
Function Analysis ◽  
Protoplast Isolation ◽  
Expression Systems ◽  
Mesophyll Protoplasts ◽  
Expression Screening ◽  
Indispensable Tool ◽  
Viable Protoplast ◽  
Gene Function Analysis

AbstractTransient expression systems in mesophyll protoplasts have been utilised in many plant species as an indispensable tool for gene function analysis and efficacious genome editing constructs. However, such a system has not been developed in Cannabis due to the recalcitrant nature of the plant to tissue culture as well as its illegal status for many years. In this study, young expanding leaves from aseptic in vitro Cannabis explants were used for protoplast isolation. Factorial designs were used to optimise variables in viable protoplast isolation and transient expression of GFP, with a range analyses performed to determine, and quantify, significantly impacting variables. Viable protoplast yields as high as 5.7 × 106 were achieved with 2.5% (w/v) Cellulase R-10, 0.3% (w/v) Macerozyme R-10 and 0.7 M mannitol, incubated for 16 h. As indicated by the transient expression of GFP, efficiency reached 23.2% with 30 μg plasmid, 50% PEG, 1 × 106 protoplasts and a transfection duration of 20 min. Application of the optimised protocol for protoplast isolation was successfully evaluated on three subsequent unrelated genotypes to highlight the robustness and broad applicability of the developed technique.

scholarly journals Efficient Delivery of dsRNA and DNA in Cultured Silkworm Cells for Gene Function Analysis Using PAMAM Dendrimers System

Insects ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 12
Author(s):  
Chenchen Lu ◽  
Zhiqing Li ◽  
Li Chang ◽  
Zhaoming Dong ◽  
Pengchao Guo ◽  
...  
Keyword(s):  
Gene Function ◽  
Culture Medium ◽  
Target Genes ◽  
High Efficiency ◽  
Function Analysis ◽  
Binding Capacity ◽  
Pamam Dendrimers ◽  
Binding Property ◽  
Gene Function Analysis

Polyamidoamine (PAMAM) dendrimers are emerging as intriguing nanovectors for nucleic acid delivery because of their unique well-defined architecture and high binding capacity, which have been broadly applied in DNA- and RNA-based therapeutics. The low-cost and high-efficiency of PAMAM dendrimers relative to traditional liposomal transfection reagents also promote their application in gene function analysis. In this study, we first investigated the potential use of a PAMAM system in the silkworm model insect. We determined the binding property of G5-PAMAM using dsRNA and DNA in vitro, and substantially achieved the delivery of dsRNA and DNA from culture medium to both silkworm BmN and BmE cells, thus leading to efficient knockdown and expression of target genes. Under treatments with different concentrations of G5-PAMAM, we evaluated its cellular cytotoxicity on silkworm cells, and the results show that G5-PAMAM had no obvious toxicity to cells. The presence of serum in the culture medium did not affect the delivery performance of DNA and dsRNA by G5-PAMAM, revealing its convenient use for various purposes. In conclusion, our data demonstrate that the PAMAM system provides a promising strategy for delivering dsRNA and DNA in cultured silkworm cells and promote its further application in individuals.

scholarly journals Towards A Genetic Model Organism: An Efficient Method For Stable Genetic Transformation of Eschscholzia Californica (Ranunculales)

2021 ◽  
Author(s):  
Dominik Lotz ◽  
Jafargholi Imani ◽  
Katrin Ehlers ◽  
Annette Becker
Keyword(s):  
Genetic Transformation ◽  
Genetic Model ◽  
Function Analysis ◽  
Model Organism ◽  
Protoplast Isolation ◽  
Model Systems ◽  
Eschscholzia Californica ◽  
Genetic Manipulations ◽  
California Poppy ◽  
Gene Function Analysis

Abstract California poppy (Eschscholzia californica) is a member of the Ranunculales, the sister order to all other eudicots and as such in a phylogenetically highly informative position. Ranunculales are known for their diverse floral morphologies and biosynthesis of many pharmaceutically relevant alkaloids. E. californica it is widely used as model system to study the conservation of flower developmental control genes. However, within the Ranunculales, options for stable genetic manipulations are rare and genetic model systems are thus difficult to establish. Here, we present a method for the efficient and stable genetic transformation via Agrobacterium tumefaciens-mediated transformation, somatic embryo induction, and regeneration of E. californica. Further, we provide a rapid method for protoplast isolation and transformation. This allows the study of gene functions in a single-cell and full plant context to enable gene function analysis and modification of alkaloid biosynthesis pathways by e.g. genome editing techniques providing important genetic resources for the genetic model organism E. californica.

scholarly journals Efficient Regeneration of Tobacco (Nicotiana tabacum L.) Plantlets from Cotyledon, Hypocotyl and Leaf Explants: An Excellent Model Plant for Gene Function Analysis

2020 ◽  
pp. 1-9
Author(s):  
Md. Shoyeb ◽  
Kanis Fatema ◽  
Md. Abdur Rauf Sarkar ◽  
Atikur Rahman ◽  
Shaikh Mizanur Rahman
Keyword(s):  
Gene Function ◽  
Callus Induction ◽  
Function Analysis ◽  
In Vitro Regeneration ◽  
Leaf Explants ◽  
Leaf Explant ◽  
Regeneration Ability ◽  
Ms Medium ◽  
Gene Function Analysis

Tobacco has been widely used as a model plant for stable and non-stable gene function analysis. Successful Agrobacterium-mediated transformation mainly depends on in vitro regeneration of tobacco plant. However, a reliable and standard regeneration protocol of tobacco using multiple explants is limited. In this study, we established a reliable and reproducible regeneration protocol of tobacco using three different explants i.e. cotyledon, hypocotyl and leaf. Preliminary, surface sterilized tobacco seeds were germinated on growth regulator free MS medium. Thereafter, in vitro germinated explants were inoculated into Murashige and Skoog [1] media supplemented with different combination and types of growth regulators for callus induction and subsequent regeneration of plantlets. It was revealed that, regeneration ability of explants is greatly influenced by type and nature of the explant. Among the three explants, higher callus induction (95%) was obtained in MS medium supplemented with 2.0 mg l-1 kinetin + 2.0 mg l-1 IAA from leaf explant. Also, leaf explant exhibited much higher regeneration ability (95%) than hypocotyl (60%) and cotyledon (45%) explants. Significantly highest number of shoots (8.0) were regenerated from leaf explants cultured on MS medium supplemented with 3.0 mg l-1 Kinetin+1.0 mg l-1 IAA compared to the other hormone combinations. Regenerated mature shoots were showed normal root after transferred onto ½ MS medium containing 0.3 mg l-1 IBA. This study will provide valuable information related to in vitro regeneration of tobacco plantlets using cotyledon, hypocotyl and leaf explants and will be used as a standard protocol for Agrobacterium-mediated transformation for gene function analysis.

scholarly journals Gene Function Analysis in the Ubiquitous Human Commensal and PathogenMalasseziaGenus

mBio ◽  
2016 ◽  
Vol 7 (6) ◽  
Author(s):  
Giuseppe Ianiri ◽  
Anna F. Averette ◽  
Joanne M. Kingsbury ◽  
Joseph Heitman ◽  
Alexander Idnurm
Keyword(s):  
Gene Function ◽  
Genetic Manipulation ◽  
Function Analysis ◽  
Exogenous Dna ◽  
Melanin Biosynthesis ◽  
Disease Symptoms ◽  
Wide Range ◽  
Transformation Methods ◽  
Gene Function Analysis

ABSTRACTThe genusMalasseziaincludes 14 species that are found on the skin of humans and animals and are associated with a number of diseases. Recent genome sequencing projects have defined the gene content of all 14 species; however, to date, genetic manipulation has not been possible for any species within this genus. Here, we develop and then optimize molecular tools for the transformation ofMalassezia furfurandMalassezia sympodialisusingAgrobacterium tumefaciensdelivery of transfer DNA (T-DNA) molecules. These T-DNAs can insert randomly into the genome. In the case ofM. furfur, targeted gene replacements were also achieved via homologous recombination, enabling deletion of theADE2gene for purine biosynthesis and of theLAC2gene predicted to be involved in melanin biosynthesis. Hence, the introduction of exogenous DNA and direct gene manipulation are feasible inMalasseziaspecies.IMPORTANCESpecies in the genusMalasseziaare a defining component of the microbiome of the surface of mammals. They are also associated with a wide range of skin disease symptoms. Many species are difficult to culturein vitro, and although genome sequences are available for the species in this genus, it has not been possible to assess gene function to date. In this study, we pursued a series of possible transformation methods and identified one that allows the introduction of DNA into two species ofMalassezia, including the ability to make targeted integrations into the genome such that genes can be deleted. This research opens a new direction in terms of now being able to analyze gene functions in this little understood genus. These tools will contribute to define the mechanisms that lead to the commensalism and pathogenicity in this group of obligate fungi that are predominant on the skin of mammals.

scholarly journals Highly Efficient Leaf Base Protoplast Isolation and Transient Expression Systems for Orchids and Other Important Monocot Crops

2021 ◽  
Vol 12 ◽  
Author(s):  
Rui Ren ◽  
Jie Gao ◽  
Dongmei Yin ◽  
Kai Li ◽  
Chuqiao Lu ◽  
...  
Keyword(s):  
Transient Expression ◽  
Transfection Efficiency ◽  
Expression System ◽  
Protoplast Isolation ◽  
Expression Systems ◽  
Protein Fusion ◽  
Leaf Base ◽  
Highly Efficient ◽  
Young Leaves ◽  
Source Materials

Versatile protoplast platforms greatly facilitate the development of modern botany. However, efficient protoplast-based systems are still challenging for numerous horticultural plants and crops. Orchids are globally cultivated ornamental and medicinal monocot plants, but few efficient protoplast isolation and transient expression systems have been developed. In this study, we established a highly efficient orchid protoplast isolation protocol by selecting suitable source materials and optimizing the enzymatic conditions, which required optimal D-mannitol concentrations (0.4–0.6 M) combined with optimal 1.2% cellulose and 0.6% macerozyme, 5 μM of 2-mercaptoethanol and 6 h digestion. Tissue- and organ-specific protoplasts were successfully isolated from young leaves [∼3.22 × 106/g fresh weight (FW)], flower pedicels (∼5.26 × 106/g FW), and young root tips (∼7.66 × 105/g FW) of Cymbidium orchids. This protocol recommends the leaf base tissues (the tender part of young leaves attached to the stem) as better source materials. High yielding viable protoplasts were isolated from the leaf base of Cymbidium (∼2.50 × 107/g FW), Phalaenopsis (1.83 × 107/g FW), Paphiopedilum (1.10 × 107/g FW), Dendrobium (8.21 × 106/g FW), Arundina (3.78 × 106/g FW) orchids, and other economically important monocot crops including maize (Zea mays) (3.25 × 107/g FW) and rice (Oryza sativa) (4.31 × 107/g FW), which showed marked advantages over previous mesophyll protoplast isolation protocols. Leaf base protoplasts of Cymbidium orchids were used for polyethylene glycol (PEG)-mediated transfection, and a transfection efficiency of more than 80% was achieved. This leaf base protoplast system was applied successfully to analyze the CsDELLA-mediated gibberellin signaling in Cymbidium orchids. We investigated the subcellular localization of the CsDELLA-green fluorescent protein fusion and analyzed the role of CsDELLA in the regulation of gibberellin to flowering-related genes via efficient transient overexpression and gene silencing of CsDELLA in Cymbidium protoplasts. This protoplast isolation and transient expression system is the most efficient based on the documented results to date. It can be widely used for cellular and molecular studies in orchids and other economically important monocot crops, especially for those lacking an efficient genetic transformation system in vivo.

scholarly journals An efficient transient expression system for gene function analysis in rose

Plant Methods ◽  
2017 ◽  
Vol 13 (1) ◽  
Author(s):  
Jun Lu ◽  
Mengjuan Bai ◽  
Haoran Ren ◽  
Jinyi Liu ◽  
Changquan Wang
Keyword(s):  
Transient Expression ◽  
Gene Function ◽  
Function Analysis ◽  
Expression System ◽  
Transient Expression System ◽  
Gene Function Analysis

‘AGRODATE’: a rapid Agrobacterium-mediated transient expression tool for gene function analysis in leaf discs

2019 ◽  
Vol 29 (2) ◽  
pp. 294-304
Author(s):  
Veda Krishnan ◽  
Joshna Jose ◽  
Monica Jolly ◽  
T. Vinutha ◽  
Raja Kumar ◽  
...  
Keyword(s):  
Transient Expression ◽  
Gene Function ◽  
Function Analysis ◽  
Leaf Discs ◽  
Gene Function Analysis

Mouse models to study von Willebrand factor structure-function relationships in vivo

2009 ◽  
Vol 29 (01) ◽  
pp. 17-20 ◽  
Author(s):  
I. Marx ◽  
I. Badirou ◽  
R. Pendu ◽  
O. Christophe ◽  
C. V. Denis
Keyword(s):  
Structure Function ◽  
Transient Expression ◽  
Von Willebrand Factor ◽  
Large Size ◽  
Mouse Hepatocytes ◽  
Function Relationship ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryVon Willebrand factor (VWF) structure-function relationship has been studied only through in vitro approaches. The VWF-deficient mouse model has been extremely useful to examine the in vivo function of VWF but does not allow a more subtle analysis of the relative importance of its different domains. However, considering the large size of VWF and its capacity to interact with various ligands in order to support platelet adhesion and aggregation, the necessity to evaluate independently these interactions appeared increasingly crucial. A recently developed technique, known as hydrodynamic injection, which allows transient expression of a transgene by mouse hepatocytes, proved very useful in this regard. Indeed, transient expression of various VWF mutants in VWF-deficient mice contributed to improve our knowledge about the role of VWF interaction with subendothelial collagens and with platelets receptors in VWF roles in haemostasis and thrombosis. These findings can provide new leads in the development of anti-thrombotic therapies.

scholarly journals Computational Methods for Structure-to-Function Analysis of Diet-Derived Catechins-Mediated Targeting of In Vitro Vasculogenic Mimicry

2021 ◽  
Vol 20 ◽  
pp. 117693512110092
Author(s):  
Abicumaran Uthamacumaran ◽  
Narjara Gonzalez Suarez ◽  
Abdoulaye Baniré Diallo ◽  
Borhane Annabi
Keyword(s):  
Machine Learning ◽  
Cancer Cells ◽  
Structural Changes ◽  
Function Analysis ◽  
Vasculogenic Mimicry ◽  
Machine Learning Algorithms ◽  
Emergent Behavior ◽  
Molecular Signature ◽  
Ovarian Cancer Cells

Background: Vasculogenic mimicry (VM) is an adaptive biological phenomenon wherein cancer cells spontaneously self-organize into 3-dimensional (3D) branching network structures. This emergent behavior is considered central in promoting an invasive, metastatic, and therapy resistance molecular signature to cancer cells. The quantitative analysis of such complex phenotypic systems could require the use of computational approaches including machine learning algorithms originating from complexity science. Procedures: In vitro 3D VM was performed with SKOV3 and ES2 ovarian cancer cells cultured on Matrigel. Diet-derived catechins disruption of VM was monitored at 24 hours with pictures taken with an inverted microscope. Three computational algorithms for complex feature extraction relevant for 3D VM, including 2D wavelet analysis, fractal dimension, and percolation clustering scores were assessed coupled with machine learning classifiers. Results: These algorithms demonstrated the structure-to-function galloyl moiety impact on VM for each of the gallated catechin tested, and shown applicable in quantifying the drug-mediated structural changes in VM processes. Conclusions: Our study provides evidence of how appropriate 3D VM compression and feature extractors coupled with classification/regression methods could be efficient to study in vitro drug-induced perturbation of complex processes. Such approaches could be exploited in the development and characterization of drugs targeting VM.

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