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Cell Systems Dec 2022To elucidate principles operating in native biological systems and to develop novel biotechnologies, synthetic biology aims to build and integrate synthetic gene... (Review)
Review
To elucidate principles operating in native biological systems and to develop novel biotechnologies, synthetic biology aims to build and integrate synthetic gene circuits within native transcriptional networks. The utility of synthetic gene circuits for cell engineering relies on the ability to control the expression of all constituent transgene components. Transgene silencing, defined as the loss of expression over time, persists as an obstacle for engineering primary cells and stem cells with transgenic cargos. In this review, we highlight the challenge that transgene silencing poses to the robust engineering of mammalian cells, outline potential molecular mechanisms of silencing, and present approaches for preventing transgene silencing. We conclude with a perspective identifying future research directions for improving the performance of synthetic gene circuits.
Topics: Animals; Transgenes; Genetic Engineering; Gene Regulatory Networks; Cell Communication; Mammals
PubMed: 36549273
DOI: 10.1016/j.cels.2022.11.005 -
Nature Biotechnology Mar 2024Inefficient knock-in of transgene cargos limits the potential of cell-based medicines. In this study, we used a CRISPR nuclease that targets a site within an exon of an...
Inefficient knock-in of transgene cargos limits the potential of cell-based medicines. In this study, we used a CRISPR nuclease that targets a site within an exon of an essential gene and designed a cargo template so that correct knock-in would retain essential gene function while also integrating the transgene(s) of interest. Cells with non-productive insertions and deletions would undergo negative selection. This technology, called SLEEK (SeLection by Essential-gene Exon Knock-in), achieved knock-in efficiencies of more than 90% in clinically relevant cell types without impacting long-term viability or expansion. SLEEK knock-in rates in T cells are more efficient than state-of-the-art TRAC knock-in with AAV6 and surpass more than 90% efficiency even with non-viral DNA cargos. As a clinical application, natural killer cells generated from induced pluripotent stem cells containing SLEEK knock-in of CD16 and mbIL-15 show substantially improved tumor killing and persistence in vivo.
Topics: CRISPR-Cas Systems; Gene Knock-In Techniques; Transgenes; Gene Editing
PubMed: 37127662
DOI: 10.1038/s41587-023-01779-8 -
Discovery Medicine Jan 2015Over the last five years, the number of clinical trials involving AAV (adeno-associated virus) and lentiviral vectors continue to increase by about 150 trials each year.... (Review)
Review
Over the last five years, the number of clinical trials involving AAV (adeno-associated virus) and lentiviral vectors continue to increase by about 150 trials each year. For continued success, AAV and lentiviral expression cassettes need to be designed to meet each disease's specific needs. This review discusses how viral vector expression cassettes can be engineered with elements to enhance target specificity and increase transgene expression. The key differences relating to target specificity between ubiquitous and tissue-specific promoters are discussed, as well as how endogenous miRNAs and their target sequences have been used to restrict transgene expression. Specifically, relevant studies indicating how cis-acting elements such as introns, WPRE, polyadenylation signals, and the CMV enhancer are highlighted to show their utility for enhancing transgene expression in gene therapy applications. All discussion bears in mind that expression cassettes have space constraints. In conclusion, this review can serve as a menu of vector genome design elements and their cost in terms of space to thoughtfully engineer viral vectors for gene therapy.
Topics: Dependovirus; Genetic Therapy; Humans; Transgenes
PubMed: 25636961
DOI: No ID Found -
Nature Biotechnology Jul 2023Generation of stable gene-edited plant lines using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) requires a...
Generation of stable gene-edited plant lines using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) requires a lengthy process of outcrossing to eliminate CRISPR-Cas9-associated sequences and produce transgene-free lines. We have addressed this issue by designing fusions of Cas9 and guide RNA transcripts to tRNA-like sequence motifs that move RNAs from transgenic rootstocks to grafted wild-type shoots (scions) and achieve heritable gene editing, as demonstrated in wild-type Arabidopsis thaliana and Brassica rapa. The graft-mobile gene editing system enables the production of transgene-free offspring in one generation without the need for transgene elimination, culture recovery and selection, or use of viral editing vectors. We anticipate that using graft-mobile editing systems for transgene-free plant production may be applied to a wide range of breeding programs and crop plants.
Topics: Gene Editing; CRISPR-Cas Systems; Plant Breeding; Plants, Genetically Modified; Transgenes
PubMed: 36593415
DOI: 10.1038/s41587-022-01585-8 -
EMBO Reports Jan 2004Green light for efficient production of transgenic farm animals (Review)
Review
Green light for efficient production of transgenic farm animals
Topics: Animals; Gene Transfer Techniques; Genetic Vectors; Lentivirus; Microinjections; Swine; Transgenes
PubMed: 14710182
DOI: 10.1038/sj.embor.7400053 -
Plant Molecular Biology Jun 2000Plant gene silencing was originally thought to be a quirk of transformation procedures, but is now recognized to be a facet of vitally important gene regulatory systems,... (Review)
Review
Plant gene silencing was originally thought to be a quirk of transformation procedures, but is now recognized to be a facet of vitally important gene regulatory systems, present in all organisms. Monocot plants, especially the grasses, play a foremost role in the agricultural economy of all nations, and their biotechnological manipulation offers great potential for both developed and developing countries. Here, we review reported instances of transgene silencing in monocots and relate the processes of transcriptional and post-transcriptional gene silencing (TGS, PTGS) in perspective to the rapidly burgeoning knowledge of these phenomena in many organisms. Recent findings include the involvement of an RNA-dependent RNA polymerase and a nuclease in PTGS systems and the close relationship between methylation and chromatin structure in TGS events.
Topics: Edible Grain; Gene Expression Regulation, Plant; Gene Silencing; Plants, Genetically Modified; Transgenes
PubMed: 10999414
DOI: 10.1023/a:1006412318311 -
Plant Molecular Biology May 2003It has been appreciated for many years that the structure of a transgene locus can have a major influence on the level and stability of transgene expression. Until... (Review)
Review
It has been appreciated for many years that the structure of a transgene locus can have a major influence on the level and stability of transgene expression. Until recently, however, it has been common practice to discard plant lines with poor or unstable expression levels in favor of those with practical uses. In the last few years, an increasing number of experiments have been carried out with the primary aim of characterizing transgene loci and studying the fundamental links between locus structure and expression. Cereals have been at the forefront of this research because molecular, genetic and cytogenetic analysis can be carried out in parallel to examine transgene loci in detail. This review discusses what is known about the structure and organization of transgene loci in cereals, both at the molecular and cytogenetic levels. In the latter case, important links are beginning to be revealed between higher order locus organization, nuclear architecture, chromatin structure and transgene expression.
Topics: Models, Genetic; Plants, Genetically Modified; Transformation, Genetic; Transgenes
PubMed: 12856933
DOI: 10.1023/a:1023941407376 -
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 -
Methods in Molecular Biology (Clifton,... 1993The exact structure of a microinjection transgene depends entirely on the aims of a particular experiment. In this context, reference to the chapters on the applications...
The exact structure of a microinjection transgene depends entirely on the aims of a particular experiment. In this context, reference to the chapters on the applications of transgenesis and technical details of transgenes described in references therein are highly recommended. However, certain general rules should be considered when designing a construct for eventual introduction into a transgenie organism. A transgene should operate like any other gene in the targeted cells of an organism, and hence, structural elements must be appropriately recognized by the transcriptional, posttranscriptional, and translational machinary of the host. A comprehensive review of the mechanisms of gene control is beyond the scope of the present volume, but the following points are indicative of the type of considerations that should contribute to the design of a transgene.
Topics: Animals; Gene Transfer Techniques; Protein Biosynthesis; Protein Processing, Post-Translational; RNA; RNA Processing, Post-Transcriptional; Transcription, Genetic; Transgenes
PubMed: 21390650
DOI: 10.1385/0-89603-245-0:115 -
Yi Chuan = Hereditas Feb 2017The traditional transgenic technologies, such as embryo microinjection, transposon-mediated integration, or lentiviral transfection, usually result in random insertions... (Review)
Review
The traditional transgenic technologies, such as embryo microinjection, transposon-mediated integration, or lentiviral transfection, usually result in random insertions of the foreign DNA into the host genome, which could have various disadvantages in the establishment of transgenic animals. Therefore, a strategy for site-specific integration of a transgene is needed to generate genetically modified animals with accurate and identical genotypes. However, the efficiency for site-specific integration of transgene is very low, which is mainly caused by two issues. The first one is the low efficiency of inducing double-strand break (DSB) at the target site of host genome in the initial process. The second one is the low efficiency of homologous recombination repair (HDR) between the target site and the donor plasmid carrying homologous arm and foreign genes. HDR is the most common mechanism for site-specific integration of a transgene. DSBs can stimulate DNA repair mainly by two competitive mechanisms, HDR and nonhomologous end joining (NHEJ). Hence, activation of HDR or inhibition of NHEJ can promote the HDR in the integration processes, thereby optimizing a specific targeting of the transgene. In this review, we summarize the recent advances in strategies for improving the site-specific integration of foreign transgene in transgenic technologies.
Topics: Animals; Animals, Genetically Modified; DNA Breaks, Double-Stranded; Recombinational DNA Repair; Transgenes
PubMed: 28242597
DOI: 10.16288/j.yczz.16-367