-
Bone Nov 2016Advancing our understanding of osteoblast biology and differentiation is critical to elucidate the pathological mechanisms responsible for skeletal diseases such as... (Review)
Review
Advancing our understanding of osteoblast biology and differentiation is critical to elucidate the pathological mechanisms responsible for skeletal diseases such as osteoporosis. Histology and histomorphometry, the classical methods to study osteoblast biology, identify osteoblasts based on their location and morphology and ability to mineralize matrix, but do not clearly define their stage of differentiation. Introduction of visual transgenes into the cells of osteoblast lineage has revolutionized the field and resulted in a paradigm shift that allowed for specific identification and isolation of subpopulations within the osteoblast lineage. Knowledge acquired from the studies based on GFP transgenes has allowed for more precise interpretation of studies analyzing targeted overexpression or deletion of genes in the osteoblast lineage. Here, we provide a condensed overview of the currently available promoter-fluorescent reporter transgenic mice that have been generated and evaluated to varying extents. We cover different stages of the lineage as transgenes have been utilized to identify osteoprogenitors, pre-osteoblasts, osteoblasts, or osteocytes. We show that each of these promoters present with advantages and disadvantages. The studies based on the use of these reporter mice have improved our understanding of bone biology. They constitute attractive models to target osteoblasts and help to understand their cell biology.
Topics: Animals; Cell Differentiation; Cell Lineage; Green Fluorescent Proteins; Humans; Luminescent Proteins; Osteoblasts; Osteocytes; Transgenes
PubMed: 27616604
DOI: 10.1016/j.bone.2016.09.004 -
Drug Delivery Nov 2017We have previously developed an efficient and safe transfection method for the kidney in mice: renal suction-mediated transfection. In this study, we verified the...
We have previously developed an efficient and safe transfection method for the kidney in mice: renal suction-mediated transfection. In this study, we verified the detailed characteristics of transgene expression and plasmid DNA (pDNA) in mice to develop therapeutic strategies and application to gene function analysis in the kidney. After naked pDNA was administered intravenously, the right kidney was immediately suctioned by a tissue suction device. We examined the spatial distribution of transgene expression and pDNA in the suctioned kidney using tissue clearing by CUBIC, Clear, and Scale SQ reagents. Spatial distribution analysis showed that pDNA was transfected into extravascular cells and sufficiently delivered to the deep renal cortex. In addition, we revealed that transgene expression occurred mainly in peritubular fibroblasts of the suctioned kidney by tissue clearing and immunohistochemistry. Next, we confirmed the periods of pDNA uptake and activation of transcription factors nuclear factor-κB and activator protein 1 by luciferase assays. Moreover, the use of a pCpG-free plasmid enabled sustained transgene expression in the suctioned kidney. In conclusion, analyses of the spatial distribution and immunostaining of the section suggest that pDNA and transgene expression occurs mainly in peritubular fibroblasts of the suctioned kidney. In addition, we clarified some factors for efficient and/or sustained transgene expression in the suctioned kidney.
Topics: Animals; DNA; Luciferases; Mice; Plasmids; Transfection; Transgenes
PubMed: 28585867
DOI: 10.1080/10717544.2017.1333171 -
Plant Physiology Mar 2022Transgene residuals in edited plants affect genetic analysis, pose off-target risks, and cause regulatory concerns. Several strategies have been developed to efficiently...
Transgene residuals in edited plants affect genetic analysis, pose off-target risks, and cause regulatory concerns. Several strategies have been developed to efficiently edit target genes without leaving any transgenes in plants. Some approaches directly address this issue by editing plant genomes with DNA-free reagents. On the other hand, DNA-based techniques require another step for ensuring plants are transgene-free. Fluorescent markers, pigments, and chemical treatments have all been employed as tools to distinguish transgenic plants from transgene-free plants quickly and easily. Moreover, suicide genes have been used to trigger self-elimination of transgenic plants, greatly improving the efficiency of isolating the desired transgene-free plants. Transgenes can also be excised from plant genomes using site-specific recombination, transposition or gene editing nucleases, providing a strategy for editing asexually produced plants. Finally, haploid induction coupled with gene editing may make it feasible to edit plants that are recalcitrant to transformation. Here, we evaluate the strengths and weaknesses of recently developed approaches for obtaining edited plants without transgene residuals.
Topics: Endonucleases; Gene Editing; Genome, Plant; Plants, Genetically Modified; Transgenes
PubMed: 34893903
DOI: 10.1093/plphys/kiab574 -
Molecular Therapy : the Journal of the... Apr 2022Recombinant adeno-associated virus (rAAV) gene therapy has the potential to transform the lives of patients with certain genetic disorders by increasing or restoring... (Review)
Review
Recombinant adeno-associated virus (rAAV) gene therapy has the potential to transform the lives of patients with certain genetic disorders by increasing or restoring function to affected tissues. Following the initial establishment of transgene expression, it is unknown how long the therapeutic effect will last, although animal and emerging human data show that expression can be maintained for more than 10 years. The durability of therapeutic response is key to long-term treatment success, especially since immune responses to rAAV vectors may prevent re-dosing with the same therapy. This review explores the non-immunological and immunological processes that may limit or improve durability and the strategies that can be used to increase the duration of the therapeutic effect.
Topics: Animals; Dependovirus; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Humans; Transgenes
PubMed: 35283274
DOI: 10.1016/j.ymthe.2022.03.004 -
International Journal of Molecular... Mar 2017Vaccination is one of the most efficient tools for disease prevention, and a continuously growing field of research. However, despite progress, we still need more... (Review)
Review
Vaccination is one of the most efficient tools for disease prevention, and a continuously growing field of research. However, despite progress, we still need more efficient and cost-effective vaccines that would improve access to those in need. In this review, we will describe the status of virus-vectored vaccine technology with a focus on adenoviral-based vaccines. Adenovirus (Ad) vaccines have proven to be efficient in military vaccinations against Ad4 and Ad7 and as highly efficient vectored vaccines against rabies. The question of how other adenovirus-based vaccines can become as efficient as the rabies vaccine is the underlying theme in this review. Here, we will first give an overview of the basic properties of vectored vaccines, followed by an introduction to the characteristics of adenoviral vectors and previously tested modifications of the vector backbone and expression cassettes, with a focus on how they can contribute to increased vaccine cost-effectiveness. Finally, we will highlight a few successful examples of research that have attempted to improve the use of adenoviral-based vaccines by improving the transgene immunogenicity.
Topics: Adenoviridae; Animals; Biotechnology; Cost-Benefit Analysis; Genetic Vectors; Humans; Immunity; Transgenes; Vaccines, Synthetic; Virus Replication
PubMed: 28420073
DOI: 10.3390/ijms18040686 -
Disease Models & Mechanisms May 2022Although a large number of mouse models have been made to study Alzheimer's disease, only a handful allow experimental control over the location or timing of the protein...
Although a large number of mouse models have been made to study Alzheimer's disease, only a handful allow experimental control over the location or timing of the protein being used to drive pathology. Other fields have used the Cre and the tamoxifen-inducible CreER driver lines to achieve precise spatial and temporal control over gene deletion and transgene expression, yet these tools have not been widely used in studies of neurodegeneration. Here, we describe two strategies for harnessing the wide range of Cre and CreER driver lines to control expression of disease-associated amyloid precursor protein (APP) in modeling Alzheimer's amyloid pathology. We show that CreER-based spatial and temporal control over APP expression can be achieved with existing lines by combining a Cre driver with a tetracycline-transactivator (tTA)-dependent APP responder using a Cre-to-tTA converter line. We then describe a new mouse line that places APP expression under direct control of Cre recombinase using an intervening lox-stop-lox cassette. Mating this allele with a CreER driver allows both spatial and temporal control over APP expression, and with it, amyloid onset. This article has an associated First Person interview with the first author of the paper.
Topics: Alleles; Amyloid beta-Protein Precursor; Animals; Humans; Integrases; Mice; Mice, Transgenic; Tetracycline; Transgenes
PubMed: 35394029
DOI: 10.1242/dmm.049330 -
Genes Dec 2021Generation of transgenic organisms by pronuclear microinjection has become a routine procedure. However, while the process of DNA integration in the genome is well... (Review)
Review
Generation of transgenic organisms by pronuclear microinjection has become a routine procedure. However, while the process of DNA integration in the genome is well understood, we still do not know much about the recombination between transgene molecules that happens in the first moments after DNA injection. Most of the time, injected molecules are joined together in head-to-tail tandem repeats-the so-called concatemers. In this review, we focused on the possible concatenation mechanisms and how they could be studied with genetic reporters tracking individual copies in concatemers. We also discuss various features of concatemers, including palindromic junctions and repeat-induced gene silencing (RIGS). Finally, we speculate how cooperation of DNA repair pathways creates a multicopy concatenated insert.
Topics: Animals; DNA; DNA End-Joining Repair; Genome; Humans; Transgenes
PubMed: 34946918
DOI: 10.3390/genes12121969 -
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 -
Viruses Dec 2020Lentiviral vectors are versatile tools for gene delivery purposes. While in the earlier versions of retroviral vectors, transgene expression was controlled by the long... (Review)
Review
Lentiviral vectors are versatile tools for gene delivery purposes. While in the earlier versions of retroviral vectors, transgene expression was controlled by the long terminal repeats (LTRs), the latter generations of vectors, including those derived from lentiviruses, incorporate internal constitutive or regulated promoters in order to regulate transgene expression. This allows to temporally and/or quantitatively control transgene expression, which is required for many applications such as for clinical applications, when transgene expression is required in specific tissues and at a specific timing. Here we review the main systems that have been developed for transgene regulated expression following lentiviral gene transfer. First, the induction of gene expression can be triggered either by external or by internal cues. Indeed, these regulated vector systems may harbor promoters inducible by exogenous stimuli, such as small molecules (e.g., antibiotics) or temperature variations, offering the possibility to tune rapidly transgene expression in case of adverse events. Second, expression can be indirectly adjusted by playing on inserted sequence copies, for instance by gene excision. Finally, synthetic networks can be developed to sense specific endogenous signals and trigger defined responses after information processing. Regulatable lentiviral vectors (LV)-mediated transgene expression systems have been widely used in basic research to uncover gene functions or to temporally reprogram cells. Clinical applications are also under development to induce therapeutic molecule secretion or to implement safety switches. Such regulatable approaches are currently focusing much attention and will benefit from the development of other technologies in order to launch autonomously controlled systems.
Topics: Animals; Cellular Reprogramming; Gene Expression; Gene Expression Regulation; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Humans; Lentivirus; Promoter Regions, Genetic; Transduction, Genetic; Transgenes
PubMed: 33322556
DOI: 10.3390/v12121427 -
Communications Biology Dec 2021Though various transgene expression switches have been adopted in a wide variety of organisms for basic and biomedical research, intrinsic obstacles of those existing...
Though various transgene expression switches have been adopted in a wide variety of organisms for basic and biomedical research, intrinsic obstacles of those existing systems, including toxicity and silencing, have been limiting their use in vertebrate transgenesis. Here we demonstrate a novel QF-based binary transgene switch (IQ-Switch) that is relatively free of driver toxicity and transgene silencing, and exhibits potent and highly tunable transgene activation by the chemical inducer tebufenozide, a non-toxic lipophilic molecule to developing zebrafish with negligible background. The interchangeable IQ-Switch makes it possible to elicit ubiquitous and tissue specific transgene expression in a spatiotemporal manner. We generated a RASopathy disease model using IQ-Switch and demonstrated that the RASopathy symptoms were ameliorated by the specific BRAF inhibitor vemurafenib, validating the therapeutic use of the gene switch. The orthogonal IQ-Switch provides a state-of-the-art platform for flexible regulation of transgene expression in zebrafish, potentially applicable in cell-based systems and other model organisms.
Topics: Animals; Animals, Genetically Modified; Gene Transfer Techniques; Genes, Switch; Transgenes; Zebrafish
PubMed: 34916605
DOI: 10.1038/s42003-021-02923-3