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Cell Reports May 2022Modern biology is increasingly reliant on optical technologies, including visualization and longitudinal monitoring of cellular processes. The major limitation here is... (Review)
Review
Modern biology is increasingly reliant on optical technologies, including visualization and longitudinal monitoring of cellular processes. The major limitation here is the availability of animal models to track the molecules and cells in their natural environment in vivo. Owing to the integrity of the studied tissue and the high stability of transgene expression throughout life, transgenic mice encoding fluorescent proteins and biosensors represent unique tools for in vivo studies in norm and pathology. We review the strategies for targeting probe expression in specific tissues, cell subtypes, or cellular compartments. We describe the application of transgenic mice expressing fluorescent proteins for tracking protein expression patterns, apoptotic events, tissue differentiation and regeneration, neurogenesis, tumorigenesis, and cell fate mapping. We overview the possibilities of functional imaging of secondary messengers, neurotransmitters, and ion fluxes. Finally, we provide the rationale and perspectives for the use of transgenic imaging probes in translational research and drug discovery.
Topics: Animals; Integrases; Mice; Mice, Transgenic; Neurogenesis; Proteins; Transgenes
PubMed: 35613592
DOI: 10.1016/j.celrep.2022.110845 -
Plant Biotechnology Journal Dec 2014Progress has been made in a 12 year's systemic study on the rice transgene flow including (i) with experiments conducted at multiple locations and years using up to 21... (Review)
Review
Progress has been made in a 12 year's systemic study on the rice transgene flow including (i) with experiments conducted at multiple locations and years using up to 21 pollen recipients, we have elucidated the patterns of transgene flow to different types of rice. The frequency to male sterile lines is 10(1) and 10(3) higher than that to O. rufipogon and rice cultivars. Wind speed and direction are the key meteorological factors affecting rice transgene flow. (ii) A regional applicable rice gene flow model is established and used to predict the maximum threshold distances (MTDs) of gene flow during 30 years in 993 major rice producing counties of southern China. The MTD0.1% for rice cultivars is basically ≤5 m in the whole region, despite climate differs significantly at diverse locations and years. This figure is particularly valuable for the commercialization and regulation of transgenic rice. (iii) The long-term fate of transgene integrated into common wild rice was investigated. Results demonstrated that the F1 hybrids of transgenic rice/O. rufipogon gradually disappeared within 3-5 years, and the Bt or bar gene was not detectable in the mixed population, suggesting the O. rufipogon may possess a strong mechanism of exclusiveness for self-protection. (iv) The flowering time isolation and a 2-m-high cloth-screen protection were proved to be effective in reducing transgene flow. We have proposed to use a principle of classification and threshold management for different types of rice.
Topics: Gene Flow; Models, Genetic; Oryza; Plants, Genetically Modified; Risk Assessment; Transgenes
PubMed: 25431202
DOI: 10.1111/pbi.12306 -
Scientific Reports Sep 2020The presence of genetically modified organisms (GMO) is commonly assessed using real-time PCR methods targeting the most common transgenic elements found in GMOs. Once...
The presence of genetically modified organisms (GMO) is commonly assessed using real-time PCR methods targeting the most common transgenic elements found in GMOs. Once the presence of GM material has been established using these screening methods, GMOs are further identified using a battery of real-time PCR methods, each being specific of one GM event and usually targeting the junction of the plant genome and of the transgenic DNA insert. If, using these specific methods, no GMO could be identified, the presence of an unauthorized GMO is suspected. In this context, the aim of this work was to develop a fast and simple method to obtain the sequence of the transgene and of its junction with plant DNA, with the presence of a screening sequence as only prior knowledge. An unauthorized GM petunia, recently found on the French market, was used as template during the development of this new molecular tool. The innovative proposed protocol is based on the circularization of fragmented DNA followed by the amplification of the transgene and of its flanking regions using long-range inverse PCR. Sequencing was performed using the Oxford Nanopore MinION technology and a bioinformatic pipeline was developed.
Topics: Computational Biology; DNA, Plant; Nanopore Sequencing; Petunia; Plants, Genetically Modified; Sequence Analysis, DNA; Transgenes
PubMed: 32934250
DOI: 10.1038/s41598-020-71614-6 -
Journal of Biosciences Sep 2019The success of viral vectors mediated gene therapy is still hampered by immunogenicity and insufficient transgene expression. Alternatively, non-viral vectors mediated... (Review)
Review
The success of viral vectors mediated gene therapy is still hampered by immunogenicity and insufficient transgene expression. Alternatively, non-viral vectors mediated gene delivery has the advantage of low immunogenicity despite showing low transgene expression. By carefully considering the advantages of each approach, hybrid vectors are currently being developed by modifying the viral vectors using non-viral biopolymers. This review provides an overview of the hybrid vectors currently being developed.
Topics: Animals; Biopolymers; Dependovirus; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Humans; Transgenes
PubMed: 31502562
DOI: No ID Found -
International Journal of Molecular... May 2020The humoral immune response elicited by adeno-associated virus (AAV)-mediated gene therapy for the treatment of mucopolysaccharidoses (MPS) poses a significant challenge... (Review)
Review
The humoral immune response elicited by adeno-associated virus (AAV)-mediated gene therapy for the treatment of mucopolysaccharidoses (MPS) poses a significant challenge to achieving therapeutic levels of transgene expression. Antibodies targeting the AAV capsid as well as the transgene product diminish the production of glycosaminoglycan (GAG)-degrading enzymes essential for the treatment of MPS. Patients who have antibodies against AAV capsid increase in number with age, serotype, and racial background and are excluded from the clinical trials at present. In addition, patients who have undergone AAV gene therapy are often excluded from the additional AAV gene therapy with the same serotype, since their acquired immune response (antibody) against AAV will limit further efficacy of treatment. Several methods are being developed to overcome this immune response, such as novel serotype design, antibody reduction by plasmapheresis and immunosuppression, and antibody evasion using empty capsids and enveloped AAV vectors. In this review, we examine the mechanisms of the anti-AAV humoral immune response and evaluate the strengths and weaknesses of current evasion strategies in order to provide an evidence-based recommendation on evading the immune response for future AAV-mediated gene therapies for MPS.
Topics: Antibodies; Capsid; Dependovirus; Genetic Therapy; Humans; Immunity, Humoral; Mucopolysaccharidoses; Transgenes
PubMed: 32414007
DOI: 10.3390/ijms21103433 -
GM Crops & Food 2014Convincing evidence has accumulated that unintended transgene escape occurs in oilseed rape, maize, cotton and creeping bentgrass. The escaped transgenes are found in... (Review)
Review
Convincing evidence has accumulated that unintended transgene escape occurs in oilseed rape, maize, cotton and creeping bentgrass. The escaped transgenes are found in variant cultivars, in wild type plants as well as in hybrids of sexually compatible species. The fact that in some cases stacked events are present that have not been planted commercially, implies unintended recombination of transgenic traits. As the consequences of this continuous transgene escape for the ecosystem cannot be reliably predicted, I propose to use more sophisticated approaches of gene technology in future. If possible GM plants should be constructed using either site-directed mutagenesis or cisgenic strategies to avoid the problem of transgene escape. In cases where a transgenic trait is needed, efficient containment should be the standard approach. Various strategies available or in development are discussed. Such a cautious approach in developing novel types of GM crops will enhance the sustainable potential of GM crops and thus increase the public trust in green gene technology.
Topics: Crops, Agricultural; Ecosystem; Gene Flow; Mutagenesis, Site-Directed; Plants, Genetically Modified; Transgenes
PubMed: 25523171
DOI: 10.4161/21645698.2014.945883 -
Cells Nov 2019is a well-established model system for basic research questions ranging from photosynthesis and organelle biogenesis, to the biology of cilia and basal bodies, to... (Review)
Review
is a well-established model system for basic research questions ranging from photosynthesis and organelle biogenesis, to the biology of cilia and basal bodies, to channelrhodopsins and photoreceptors. More recently, has also been recognized as a suitable host for the production of high-value chemicals and high-value recombinant proteins. However, basic and applied research have suffered from the inefficient expression of nuclear transgenes. The combined efforts of the community over the past decades have provided insights into the mechanisms underlying this phenomenon and have resulted in mutant strains defective in some silencing mechanisms. Moreover, many insights have been gained into the parameters that affect nuclear transgene expression, like promoters, introns, codon usage, or terminators. Here I critically review these insights and try to integrate them into design suggestions for the construction of nuclear transgenes that are to be expressed at high levels.
Topics: Cell Nucleus; Chlamydomonas reinhardtii; Cloning, Molecular; Gene Expression Regulation, Plant; Gene Silencing; Genes, Plant; Genome, Plant; Transformation, Genetic; Transgenes
PubMed: 31795196
DOI: 10.3390/cells8121534 -
Scientific Reports Mar 2017Somatic cells can be reprogrammed to induced hepatocyte-like cells (iHeps) by overexpressing certain defined factors in direct reprogramming techniques. Of the various...
Somatic cells can be reprogrammed to induced hepatocyte-like cells (iHeps) by overexpressing certain defined factors in direct reprogramming techniques. Of the various methods to deliver genes into cells, typically used genome-integrating viral vectors are associated with integration-related adverse events such as mutagenesis, whereas non-integrating viral vectors have low efficiency, making viral vectors unsuitable for clinical application. Therefore, we focused on developing a transposon system to establish a non-viral reprogramming method. Transposons are unique DNA elements that can be integrated into and removed from chromosomes. PiggyBac, a type of transposon, has high transduction efficiency and cargo capacity, and the integrated transgene can be precisely excised in the presence of transposase. This feature enables the piggyBac vector to achieve efficient transgene expression and a transgene-free state, thus making it a promising method for cell reprogramming. Here, we attempted to utilize the piggyBac transposon system to generate iHeps by integrating a transgene consisting of Hnf4a and Foxa3, and successfully obtained functional iHeps. We then demonstrated removal of the transgene to obtain transgene-free iHeps, which still maintained hepatocyte functions. This non-viral, transgene-free reprogramming method using the piggyBac vector may facilitate clinical applications of iHeps in upcoming cell therapy.
Topics: Cellular Reprogramming; DNA Transposable Elements; Gene Transfer Techniques; Genetic Vectors; Hepatocytes; Humans; Transgenes; Transposases
PubMed: 28295042
DOI: 10.1038/srep44498 -
The CRISPR Journal Apr 2023Microinjected transgenes, both large and small, are known to insert randomly into the mouse genome. Traditional methods of mapping a transgene are challenging, thus...
Microinjected transgenes, both large and small, are known to insert randomly into the mouse genome. Traditional methods of mapping a transgene are challenging, thus complicating breeding strategies and accurate interpretation of phenotypes, particularly when a transgene disrupts critical coding or noncoding sequences. As the vast majority of transgenic mouse lines remain unmapped, we developed CRISPR-Cas9 Long-Read Sequencing (CRISPR-LRS) to ascertain transgene integration loci. This novel approach mapped a wide size range of transgenes and uncovered more complex transgene-induced host genome re-arrangements than previously appreciated. CRISPR-LRS offers a facile, informative approach to establish robust breeding practices and will enable researchers to study a gene without confounding genetic issues. Finally, CRISPR-LRS will find utility in rapidly and accurately interrogating gene/genome editing fidelity in experimental and clinical settings.
Topics: Animals; Mice; Gene Editing; CRISPR-Cas Systems; Transgenes; Genome; Mice, Transgenic
PubMed: 37071672
DOI: 10.1089/crispr.2022.0099 -
PloS One 2013The ability to achieve precisely tailored activation and inactivation of gene expression represents a critical utility for vertebrate model organisms. In this regard,...
The ability to achieve precisely tailored activation and inactivation of gene expression represents a critical utility for vertebrate model organisms. In this regard, Cre and other site-specific DNA recombinases have come to play a central role in achieving temporally regulated and cell type-specific genetic manipulation. In zebrafish, both Cre and Flp recombinases have been applied for inducible activation, inactivation and inversion of inserted genomic elements. Here we describe the addition of Dre, a heterospecific Cre-related site-specific recombinase, to the zebrafish genomic toolbox. Combining Dre-based recombination in zebrafish with established Cre/lox technology, we have established an effective strategy for transgene activation and inactivation using lox and rox (TAILOR). Using stable transgenic lines expressing tamoxifen-inducible CreER(T2) and RU486-inducible DrePR fusions, we demonstrate that Cre and Dre retain non-overlapping specificities for their respective lox and rox target sites in larval zebrafish, and that their combinatorial and sequential activation can achieve precisely timed transgene activation and inactivation. In addition to TAILOR, the successful application of Dre/rox technology in zebrafish will facilitate a variety of additional downstream genetic applications, including sequential lineage labeling, complex genomic rearrangements and the precise temporal and spatial control of gene expression through the intersection of partially overlapping promoter activities.
Topics: Animals; Escherichia coli Proteins; Gene Expression Regulation; Gene Transfer Techniques; Integrases; Microscopy, Confocal; Mifepristone; Recombinases; Substrate Specificity; Transgenes; Zebrafish
PubMed: 24391998
DOI: 10.1371/journal.pone.0085218