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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 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 -
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 -
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 -
Frontiers in Immunology 2023Use of adeno-associated virus (AAV) vectors is complicated by host immune responses that can limit transgene expression. Recent clinical trials using AAV vectors to...
INTRODUCTION
Use of adeno-associated virus (AAV) vectors is complicated by host immune responses that can limit transgene expression. Recent clinical trials using AAV vectors to deliver HIV broadly neutralizing antibodies (bNAbs) by intramuscular administration resulted in poor expression with anti-drug antibodies (ADA) responses against the bNAb.
METHODS
Here we compared the expression of, and ADA responses against, an anti-SIV antibody ITS01 when delivered by five different AAV capsids. We first evaluated ITS01 expression from AAV vectors three different 2A peptides. Rhesus macaques were selected for the study based on preexisiting neutralizing antibodies by evaluating serum samples in a neutralization assay against the five capsids used in the study. Macaques were intramuscularly administered AAV vectors at a 2.5x10^12 vg/kg over eight administration sites. ITS01 concentrations and anti-drug antibodies (ADA) were measured by ELISA and a neutralization assay was conducted to confirm antibody potency.
RESULTS
We observed that ITS01 expressed three-fold more efficiently in mice from AAV vectors in which heavy and light-chain genes were separated by a P2A ribosomal skipping peptide, compared with those bearing F2A or T2A peptides. We then measured the preexisting neutralizing antibody responses against three traditional AAV capsids in 360 rhesus macaques and observed that 8%, 16%, and 42% were seronegative for AAV1, AAV8, and AAV9, respectively. Finally, we compared ITS01 expression in seronegative macaques intramuscularly transduced with AAV1, AAV8, or AAV9, or with the synthetic capsids AAV-NP22 or AAV-KP1. We observed at 30 weeks after administration that AAV9- and AAV1-delivered vectors expressed the highest concentrations of ITS01 (224 µg/mL, n=5, and 216 µg/mL, n=3, respectively). The remaining groups expressed an average of 35-73 µg/mL. Notably, ADA responses against ITS01 were observed in six of the 19 animals. Lastly, we demonstrated that the expressed ITS01 retained its neutralizing activity with nearly the same potency of purified recombinant protein.
DISCUSSION
Overall, these data suggest that the AAV9 capsid is a suitable choice for intramuscular expression of antibodies in nonhuman primates.
Topics: Animals; Mice; Macaca mulatta; Dependovirus; Antibodies, Neutralizing; Transgenes; Capsid
PubMed: 37153616
DOI: 10.3389/fimmu.2023.1105617 -
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 -
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 -
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 -
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