Agrobacterium strains transfer a single-strand form of T-DNA (T-strands) and Virulence (Vir) effector proteins to plant cells. Following transfer, T-strands likely form complexes with Vir and plant ...proteins that traffic through the cytoplasm and enter the nucleus. T-strands may subsequently randomly integrate into plant chromosomes and permanently express encoded transgenes, a process known as stable transformation. The molecular processes by which T-strands integrate into the host genome remain unknown. Although integration resembles DNA repair processes, the requirement of known DNA repair pathways for integration is controversial. The configuration and genomic position of integrated T-DNA molecules likely affect transgene expression, and control of integration is consequently important for basic research and agricultural biotechnology applications. This article reviews our current knowledge of the process of T-DNA integration and proposes ways in which this knowledge may be manipulated for genome editing and synthetic biology purposes.
Abstract
As millions of seeds are produced from a breeding line, the long-term stability of transgene expression is vital for commercial-scale production of seeds with transgenic traits. Transgenes ...can be silenced by epigenetic mechanisms, but reactivation of expression can occur as a result of treatment with chromatin modification inhibitors such as 5-azacytidine, from stress such as heat or UV-B, or in mutants that have acquired a defect in gene silencing. Previously, we targeted a gfp reporter gene into the tobacco (Nicotiana tabacum) genome by site-specific recombination but still found some silenced lines among independent integration events. One such line also had a second random copy and both copies showed DNA hypermethylation. To test whether removing the second copy would reactivate gfp expression, two T1 plants were backcrossed to the wild type. Whereas the silenced status was maintained in the progenies from one backcross, spontaneous partial reactivation of gfp expression was found among progenies from a second backcross. However, this reactivation did not correlate with loss of the second random copy or with a significant change in the pattern or amount of DNA hypermethylation. This finding supports the suggestion that gene reactivation does not necessarily involve loss of DNA homology or methylation.
A site-specific silenced gfp transgene associated with a second gfp copy and DNA hypermethylation can be reactivated independent of a change in DNA methylation or transgene copy number.
Coxsackievirus A21 (CVA21) is a naturally occurring RNA virus that, in preclinical studies and clinical trials, has demonstrated promising potential in treating a range of malignancies. Other ...oncolytic viruses, such as adenovirus, vesicular stomatitis virus, herpesvirus, and vaccinia virus, all can be engineered to carry one or more transgenes for various purposes, including immune modulation, virus attenuation, and induction of apoptosis of tumor cells. However, it remained unknown whether CVA21 can express therapeutic or immunomodulatory payloads due to its small size and high mutation rate. Using reverse genetics techniques, we demonstrated that a transgene encoding a truncated green fluorescent protein (GFP) of up to 141 amino acids (aa) can be successfully carried in the 5' end of the coding region. Furthermore, a chimeric virus carrying an eel fluorescent protein, UnaG (139 aa), was also made and shown to be stable, and it maintained efficient tumor cell-killing activity. Similar to other oncolytic viruses, the likelihood of delivering CVA21 by the intravenous route is low due to issues like blood absorption, neutralizing antibodies, and liver clearance. To address this problem, we designed the CVA21 cDNA under the control of a weak RNA polymerase II promoter, and subsequently, a stable cell pool in 293T cells was made by integrating the resulting CVA21 cDNA into the cell genome. We showed that the cells are viable and able to persistently generate rCVA21
. The carrier cell approach described here may pave the way to designing new cell therapy strategies by arming with oncolytic viruses.
As a naturally occurring virus, coxsackievirus A21 is a promising oncolytic virotherapy modality. In this study, we first used reverse genetics to determine whether A21 can stably carry transgenes and found that it could express up to 141 amino acids of foreign GFP. The chimeric virus carrying another fluorescent eel protein UnaG (139 amino acids) gene also appeared to be stable over at least 7 passages. Our results provided guidance on how to select and engineer therapeutic payloads for future A21 anticancer research. Second, the challenges of delivering oncolytic viruses by the intravenous route hamper the broader use of oncolytic viruses in the clinic. Here, we used A21 to show that cells could be engineered to stably carry and persistently release the virus by harboring the viral cDNA in the genome. The approach we presented here may pave a new way for oncolytic virus administration using cells as carriers.
Achieving gain‐of‐function phenotypes without inserting foreign DNA is an important challenge for plant biotechnologists. Here, we show that a gene can be brought under the control of a promoter from ...an upstream gene by deleting the intervening genomic sequence using dual‐guide CRISPR/Cas9. We fuse the promoter of a nonessential photosynthesis‐related gene to DIACYLGLYCEROL ACYLTRANSFERASE 2 (DGAT2) in the lipase‐deficient sugar‐dependent 1 mutant of Arabidopsis thaliana to drive ectopic oil accumulation in leaves. DGAT2 expression is enhanced more than 20‐fold and the triacylglycerol content increases by around 30‐fold. This deletion strategy offers a transgene‐free route to engineering traits that rely on transcriptional gain‐of‐function, such as producing high lipid forage to increase the productivity and sustainability of ruminant farming.
Our study demonstrates a method to create a transcriptional gain‐of‐function without inserting foreign DNA into an organism. We bring a gene under the control of a promoter from an upstream gene by deleting the intervening genomic region using CRISPR/Cas9. We use this approach to engineer plant leaves to accumulate oil.
The commercial application of genetically modified plants has been seriously impeded by public concern surrounding the potential risks posed by such plants to the ecosystem and human health. ...Previously, we have developed a 'pollen- and seed-specific Gene Deletor' system that automatically excised all transgenes from the pollen and seeds of greenhouse-grown transgenic
. In this study, we conducted seven field experiments over three consecutive years to evaluate the stability of transgene excision under field conditions. Our results showed that transgenes were stably excised from transgenic
under field conditions with 100% efficiency. The stability of transgene excision was confirmed based on PCR, as well as the GUS staining patterns of various organs (roots, leaves, petiole, stem, flower, fruit, and seeds) from transgenic
. In six transgenic lines (D4, D10, D31, D56, and D43), the transgenes were stably deleted in the T0 and T1 generations. Thus, the 'Gene Deletor' system is an efficient and reliable method to reduce pollen- and seed-mediated unintentional gene flow. This system might help to alleviate the food safety concerns associated with transgenic crops.
Among the most widely grown cereal crops is maize, which is a staple food for millions of people worldwide. It is primarily used for human consumption in various forms, animal feed, and industrial ...applications. In many countries like Mexico, Africa, and South America, it is the main source of calories in their daily diet, making it crucial for food security. Many nations worldwide are more at risk of drought as global warming continues to accelerate. One of the major hurdles to food production in the twenty-first century and a serious threat to our present and future food security is a water crisis. Crop failure due to water scarcity can put millions of lives at risk. Along with traditional breeding, transgenic approaches are an essential tool in modern plant breeding. They allow the introduction of beneficial genes from other organisms or within the same organism to improve plant characteristics. This review focuses on specific genes that are stably expressed and tested for drought tolerance in maize. Several genes have been identified as potential targets for improving drought tolerance in maize. Although mechanisms of target genes overlap to some extent, we attempted to divide the selected research articles according to the mechanism of the targeted gene into categories and reviewed them.
Hepatocyte-restricted, AAV-mediated gene transfer is being used to provide sustained, tolerogenic transgene expression in gene therapy. However, given the episomal status of the AAV genome, this ...approach cannot be applied to pediatric disorders when hepatocyte proliferation may result in significant loss of therapeutic efficacy over time. In addition, many multi-systemic diseases require widespread expression of the therapeutic transgene that, when provided with ubiquitous or tissue-specific non-hepatic promoters, often results in anti-transgene immunity. Here we have developed tandem promoter monocistronic expression cassettes that, packaged in a single AAV, provide combined hepatic and extra-hepatic tissue-specific transgene expression and prevent anti-transgene immunity. We validated our approach in infantile Pompe disease, a prototype disease caused by lack of the ubiquitous enzyme acid-alpha-glucosidase (GAA), presenting multi-systemic manifestations and detrimental anti-GAA immunity. We showed that the use of efficient tandem promoters prevents immune responses to GAA following systemic AAV gene transfer in immunocompetent Gaa-/- mice. Then we demonstrated that neonatal gene therapy with either AAV8 or AAV9 in Gaa-/- mice resulted in persistent therapeutic efficacy when using a tandem liver-muscle promoter (LiMP) that provided high and persistent transgene expression in non-dividing extra-hepatic tissues. In conclusion, the tandem promoter design overcomes important limitations of AAV-mediated gene transfer and can be beneficial when treating pediatric conditions requiring persistent multi-systemic transgene expression and prevention of anti-transgene immunity.
•AAV constructs express transgenes in CNS cells, but microglia present challenges.•Novel, genetically modified AAV capsids show improved microglial transduction.•Myeloid-specific promoters in AAV ...constructs fail to prevent off-target expression.•Microscopy and PCR controls may minimize false positive AAV transgene detection.
Microglia play a central role in the etiology of many neuropathologies. Transgenic tools are a powerful experiment approach to gain reliable and specific control over microglia function. Adeno-associated virus (AAVs) vectors are already an indispensable tool in neuroscience research. Despite ubiquitous use of AAVs and substantial interest in the role of microglia in the study of central nervous system (CNS) function and disease, transduction of microglia using AAVs is seldom reported. This review explores the challenges and advancements made in using AAVs for expressing transgenes in microglia. First, we will examine the functional anatomy of the AAV capsid, which will serve as a basis for subsequent discussions of studies exploring the relationship between capsid mutations and microglia transduction efficacy. After outlining the functional anatomy of AAVs, we will consider the experimental evidence demonstrating AAV-mediated transduction of microglia and microglia-like cell lines followed by an examination of the most promising experimental approaches identified in the literature. Finally, technical limitations will be considered in future applications of AAV experimental approaches.
Abstract Plants respond to biotic and abiotic stress by activating and interacting with multiple defense pathways, allowing for an efficient global defense response. RNA silencing is a conserved ...mechanism of regulation of gene expression directed by small RNAs important in acquired plant immunity and especially virus and transgene repression. Several RNA silencing pathways in plants are crucial to control developmental processes and provide protection against abiotic and biotic stresses as well as invasive nucleic acids such as viruses and transposable elements. Various notable studies have shed light on the genes, small RNAs, and mechanisms involved in plant RNA silencing. However, published research on the potential interactions between RNA silencing and other plant stress responses is limited. In the present study, we tested the hypothesis that spreading and maintenance of systemic post-transcriptional gene silencing (PTGS) of a GFP transgene are associated with transcriptional changes that pertain to non-RNA silencing-based stress responses. To this end, we analyzed the structure and function of the photosynthetic apparatus and conducted whole transcriptome analysis in a transgenic line of Nicotiana benthamiana that spontaneously initiates transgene silencing, at different stages of systemic GFP-PTGS. In vivo analysis of chlorophyll a fluorescence yield and expression levels of key photosynthetic genes indicates that photosynthetic activity remains unaffected by systemic GFP-PTGS. However, transcriptomic analysis reveals that spreading and maintenance of GFP-PTGS are associated with transcriptional reprogramming of genes that are involved in abiotic stress responses and pattern- or effector-triggered immunity-based stress responses. These findings suggest that systemic PTGS may affect non-RNA-silencing-based defense pathways in N. benthamiana , providing new insights into the complex interplay between different plant stress responses.
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