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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 -
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 -
Cell Jul 2018Modern genetic approaches are powerful in providing access to diverse cell types in the brain and facilitating the study of their function. Here, we report a large set...
Modern genetic approaches are powerful in providing access to diverse cell types in the brain and facilitating the study of their function. Here, we report a large set of driver and reporter transgenic mouse lines, including 23 new driver lines targeting a variety of cortical and subcortical cell populations and 26 new reporter lines expressing an array of molecular tools. In particular, we describe the TIGRE2.0 transgenic platform and introduce Cre-dependent reporter lines that enable optical physiology, optogenetics, and sparse labeling of genetically defined cell populations. TIGRE2.0 reporters broke the barrier in transgene expression level of single-copy targeted-insertion transgenesis in a wide range of neuronal types, along with additional advantage of a simplified breeding strategy compared to our first-generation TIGRE lines. These novel transgenic lines greatly expand the repertoire of high-precision genetic tools available to effectively identify, monitor, and manipulate distinct cell types in the mouse brain.
Topics: Animals; Brain; Calcium; Cell Line; Gene Knockout Techniques; Genes, Reporter; In Situ Hybridization, Fluorescence; Light; Mice; Mice, Transgenic; Microscopy, Fluorescence; Neurons; Optogenetics; RNA, Untranslated; Transgenes
PubMed: 30007418
DOI: 10.1016/j.cell.2018.06.035 -
Proceedings of the National Academy of... Aug 2023Tissue macrophages, including microglia, are notoriously resistant to genetic manipulation. Here, we report the creation of Adeno-associated viruses (AAV) variants that...
Tissue macrophages, including microglia, are notoriously resistant to genetic manipulation. Here, we report the creation of Adeno-associated viruses (AAV) variants that efficiently and widely transduce microglia and tissue macrophages in vivo following intravenous delivery, with transgene expression of up to 80%. We use this technology to demonstrate manipulation of microglia gene expression and microglial ablation, thereby providing invaluable research tools for the study of these important cells.
Topics: Dependovirus; Microglia; Capsid; Transgenes; Macrophages
PubMed: 37603759
DOI: 10.1073/pnas.2302997120 -
Genome Research Mar 2019Transgenesis has been a mainstay of mouse genetics for over 30 yr, providing numerous models of human disease and critical genetic tools in widespread use today....
Transgenesis has been a mainstay of mouse genetics for over 30 yr, providing numerous models of human disease and critical genetic tools in widespread use today. Generated through the random integration of DNA fragments into the host genome, transgenesis can lead to insertional mutagenesis if a coding gene or an essential element is disrupted, and there is evidence that larger scale structural variation can accompany the integration. The insertion sites of only a tiny fraction of the thousands of transgenic lines in existence have been discovered and reported, due in part to limitations in the discovery tools. Targeted locus amplification (TLA) provides a robust and efficient means to identify both the insertion site and content of transgenes through deep sequencing of genomic loci linked to specific known transgene cassettes. Here, we report the first large-scale analysis of transgene insertion sites from 40 highly used transgenic mouse lines. We show that the transgenes disrupt the coding sequence of endogenous genes in half of the lines, frequently involving large deletions and/or structural variations at the insertion site. Furthermore, we identify a number of unexpected sequences in some of the transgenes, including undocumented cassettes and contaminating DNA fragments. We demonstrate that these transgene insertions can have phenotypic consequences, which could confound certain experiments, emphasizing the need for careful attention to control strategies. Together, these data show that transgenic alleles display a high rate of potentially confounding genetic events and highlight the need for careful characterization of each line to assure interpretable and reproducible experiments.
Topics: Animals; Cells, Cultured; Genomic Structural Variation; Genotyping Techniques; Mice; Mice, Transgenic; Mutagenesis, Insertional; Nucleic Acid Amplification Techniques; Phenotype; Recombination, Genetic; Transgenes
PubMed: 30659012
DOI: 10.1101/gr.233866.117 -
Plant Physiology Mar 2022An integrated transgene-free multiplex gene-editing toolkit based on the Transgene Killer CRISPR technology greatly saves labor, time, and cost.
An integrated transgene-free multiplex gene-editing toolkit based on the Transgene Killer CRISPR technology greatly saves labor, time, and cost.
Topics: CRISPR-Cas Systems; Gene Editing; Transgenes
PubMed: 34893900
DOI: 10.1093/plphys/kiab573 -
Mammalian Genome : Official Journal of... Mar 2022Recombinase alleles and transgenes can be used to facilitate spatio-temporal specificity of gene disruption or transgene expression. However, the versatility of this in... (Review)
Review
Recombinase alleles and transgenes can be used to facilitate spatio-temporal specificity of gene disruption or transgene expression. However, the versatility of this in vivo recombination system relies on having detailed and accurate characterization of recombinase expression and activity to enable selection of the appropriate allele or transgene. The CrePortal ( http://www.informatics.jax.org/home/recombinase ) leverages the informatics infrastructure of Mouse Genome Informatics to integrate data from the scientific literature, direct data submissions from the scientific community at-large, and from major projects developing new recombinase lines and characterizing recombinase expression and specificity patterns. Searching the CrePortal by recombinase activity or specific recombinase gene driver provides users with a recombinase alleles and transgenes activity tissue summary and matrix comparison of gene expression and recombinase activity with links to generation details, a recombinase activity grid, and associated phenotype annotations. Future improvements will add cell type-based activity annotations. The CrePortal provides a comprehensive presentation of recombinase allele and transgene data to assist researchers in selection of the recombinase allele or transgene based on where and when recombination is desired.
Topics: Alleles; Animals; Integrases; Mice; Mice, Transgenic; Recombinases; Transgenes
PubMed: 34482425
DOI: 10.1007/s00335-021-09909-w -
Frontiers in Immunology 2018Gene therapy aims to replace a defective or a deficient protein at therapeutic or curative levels. Improved vector designs have enhanced safety, efficacy, and delivery,... (Review)
Review
Gene therapy aims to replace a defective or a deficient protein at therapeutic or curative levels. Improved vector designs have enhanced safety, efficacy, and delivery, with potential for lasting treatment. However, innate and adaptive immune responses to the viral vector and transgene product remain obstacles to the establishment of therapeutic efficacy. It is widely accepted that endogenous regulatory T cells (Tregs) are critical for tolerance induction to the transgene product and in some cases the viral vector. There are two basic strategies to harness the suppressive ability of Tregs: induction of adaptive Tregs specific to the introduced gene product and concurrent administration of autologous, expanded Tregs. The latter may be polyclonal or engineered to direct specificity to the therapeutic antigen. Recent clinical trials have advanced adoptive immunotherapy with Tregs for the treatment of autoimmune disease and in patients receiving cell transplants. Here, we highlight the potential benefit of combining gene therapy with Treg adoptive transfer to achieve a sustained transgene expression. Furthermore, techniques to engineer antigen-specific Treg cell populations, either through reprogramming conventional CD4 T cells or transferring T cell receptors with known specificity into polyclonal Tregs, are promising in preclinical studies. Thus, based upon these observations and the successful use of chimeric (IgG-based) antigen receptors (CARs) in antigen-specific effector T cells, different types of CAR-Tregs could be added to the repertoire of inhibitory modalities to suppress immune responses to therapeutic cargos of gene therapy vectors. The diverse approaches to harness the ability of Tregs to suppress unwanted immune responses to gene therapy and their perspectives are reviewed in this article.
Topics: Animals; Antigen-Presenting Cells; Autoimmune Diseases; Genetic Therapy; Genetic Vectors; Humans; Lentivirus; Models, Immunological; T-Lymphocytes, Regulatory; Transgenes
PubMed: 29616042
DOI: 10.3389/fimmu.2018.00554 -
Plant Biotechnology Journal Feb 2016The demand for crops requiring increasingly complex combinations of transgenes poses unique challenges for transgenic trait deployment. Future value-adding traits such... (Review)
Review
The demand for crops requiring increasingly complex combinations of transgenes poses unique challenges for transgenic trait deployment. Future value-adding traits such as those associated with crop performance are expected to involve multiple transgenes. Random integration of transgenes not only results in unpredictable expression and potential unwanted side effects but stacking multiple, randomly integrated, independently segregating transgenes creates breeding challenges during introgression and product development. Designed nucleases enable the creation of targeted DNA double-strand breaks at specified genomic locations whereby repair can result in targeted transgene integration leading to precise alterations in DNA sequences for plant genome editing, including the targeting of a transgene to a genomic locus that supports high-level and stable transgene expression without interfering with resident gene function. In addition, targeted DNA integration via designed nucleases allows for the addition of transgenes into previously integrated transgenic loci to create stacked products. The currently reported frequencies of independently generated transgenic events obtained with site-specific transgene integration without the aid of selection for targeting are very low. A modular, positive selection-based gene targeting strategy has been developed involving cassette exchange of selectable marker genes which allows for targeted events to be preferentially selected, over multiple cycles of sequential transformation. This, combined with the demonstration of intragenomic recombination following crossing of transgenic events that contain stably integrated donor and target DNA constructs with nuclease-expressing plants, points towards the future of trait stacking that is less dependent on high-efficiency transformation.
Topics: Biotechnology; Endonucleases; Plants, Genetically Modified; Quantitative Trait, Heritable; Recombination, Genetic; Transgenes
PubMed: 26332789
DOI: 10.1111/pbi.12457