Timing Of Transgene Expression Research Articles

Parsley ubiquitin promoter displays higher activity than the CaMV 35S promoter and the chrysanthemum actin 2 promoter for productive, constitutive, and durable expression of a transgene in Chrysanthemum morifolium.

The chrysanthemum (Chrysanthemum morifolium) is one of the most popular ornamental plants in the world. Genetic transformation is a promising tool for improving traits, editing genomes, and studying plant physiology. Promoters are vital components for efficient transformation, determining the level, location, and timing of transgene expression. The cauliflower mosaic virus (CaMV) 35S promoter is most frequently used in dicotyledonous plants but is less efficient in chrysanthemums than in tobacco or torenia plants. Previously, we used the parsley ubiquitin (PcUbi) promoter in chrysanthemums for the first time and analyzed its activity in transgenic calli. To expand the variety of constitutive promoters in chrysanthemums, we cloned the upstream region of the actin 2 (CmACT2) gene and compared its promoter activity with the 35S and PcUbi promoters in several organs, as well as its durability for long-term cultivation. The CmACT2 promoter has higher activity than the 35S promoter in calli but is less durable. The PcUbi promoter has the highest activity not only in calli but also in leaves, ray florets, and disk florets, and retains its activity after long-term cultivation. In conclusion, we have provided useful information and an additional type of promoter available for transgene expression in chrysanthemums.

Pseudotyped adeno‐associated viral vector tropism and transduction efficiencies in murine wound healing

Cell specific gene transfer and sustained transgene expression are goals of cutaneous gene therapy for tissue repair and regeneration. Adeno-associated virus serotype 2 (AAV2/2) mediated gene transfer to the skin results in stable transgene expression in the muscle fascicles of the panniculus carnosus in mice, with minimal gene transfer to the dermal or epidermal elements. We hypothesized that pseudotyped AAV vectors may have a unique and characteristic tropism and transduction efficiency profile for specific cells in the cutaneous wounds. We compared transduction efficiencies of cells in the epidermis, cells in the dermis, and the fascicles of the panniculus carnosus by AAV2/2 and three pseudotyped AAV vectors, AAV2/5, AAV2/7, and AAV2/8 in a murine excisional wound model. AAV2/5 and AAV2/8 result in significantly enhanced transduction of cells both in the epidermis and the dermis compared to AAV2/2. AAV2/5 transduces both the basilar and supra-basilar keratinocytes. In contrast, AAV2/8 transduces mainly supra-basilar keratinocytes. Both AAV2/7 and AAV2/8 result in more efficient gene transfer to the muscular panniculus carnosus compared to AAV2/2. The capsid of the different pseudotyped AAV vectors produces distinct tropism and efficiency profiles in the murine wound healing model. Both AAV2/5 and AAV2/8 administration result in significantly enhanced gene transfer. To further characterize cell specific transduction and tropism profiles of the AAV pseudotyped vectors, we performed in vitro experiments using human and mouse primary dermal fibroblasts. Our data demonstrate that pseudotyping strategy confers a differential transduction of dermal fibroblasts, with higher transduction of both human and murine cells by AAV2/5 and AAV2/8 at early and later time points. At later time points, AAV2/2 demonstrates increased transduction. Interestingly, AAV2/8 appears to be more efficacious in transducing human cells as compared to AAV2/5. The pseudotype-specific pattern of transduction and tropism observed both in vivo and in vitro suggests that choice of AAV vectors should be based on the desired target cell and the timing of transgene expression in wound healing for gene transfer therapy in dermal wounds.

In vivo molecular imaging characterizes pulmonary gene expression in experimental lung transplantation

Abstract Introduction: Gene therapy is effective in preventing ischemia-reperfusion injury and allograft rejection after experimental lung transplantation. Currently, no means exist to noninvasively determine the distribution, magnitude and timing of transgene expression after transplantation. This study utilizes a novel PET reporter gene system to characterize transgene expression after experimental lung transplantation. Methods: Experiments utilized a rodent orthotopic left lung transplant model. Donors underwent endotracheal transfection 24 hours before harvest with 5x1010 VPs of adenovirus encoding a fusion gene of thymidine kinase (TK, PET reporter gene) and green fluorescent protein. PET images were obtained in 4 groups of recipients: 24 hours postoperatively with and without I/R injury, and 4 days postoperatively with and without acute rejection. Rats which underwent transfection alone served as controls. Excised lungs were assessed for gamma counter radioactivity and TK activity. Results: Transplanted lungs displayed strong and linear correlation between PET signals and gamma counter radioactivity (r2 = 0.82, p Conclusions: PET imaging is a sensitive and quantitative method for characterizing pulmonary transgene expression in experimental lung transplantation. Characterization of transgene expression is not adversely affected by I/R injury or graft rejection.

Strategy of liver-directed gene therapy: present status and future prospects.

The liver is particularly amenable to gene therapy as it is the site of many metabolic diseases and malignancies. Thus, liver-directed gene therapy is being actively pursued and developed as a method of treatment for various liver diseases. Strategies of liver-directed gene therapy include drug delivery to the liver, compensation of the defective gene(s), anti-tumor activity, anti-viral therapy, and immunomodulation. The strategy chosen for liver-directed gene therapy depends on the genetic basis of the disease. Many aspects are key factors to the success of the chosen strategy: intervention of genes, efficient gene delivery system, stable transgene expression, transgene regulation, target cell transfection, and timing of transgene expression. Several tactics can be used to overcome problems in the above, and these include the use of a gene switch to exogenously regulate transgene expression, targeting at the transcriptional level, circumvention of the immune response (as in the use of adenovirus vector to achieve long-term correction of genetic diseases), and genetically engineered antibodies in gene transfer. At the present rate of research activity and development, gene therapies may soon be more efficient than current standard treatments for some liver diseases.