Direct Transgene Expression Research Articles

Characterization and functional analysis of novel α-bisabolol synthase (MrBAS) promoter from Matricaria recutita

Matricaria recutita is widely used in industry and as a medicinal plant because it contains α-bisabolol. Alpha-bisabolol has broad application prospects due to its healthy function and medical value. The activity of the α-bisabolol synthase (MrBAS) promoter determines the expression of the MrBAS gene, which in turn influences the synthesis and accumulation of α-bisabolol. However, the activity and tissue specificity of the MrBAS promoter have not yet been characterized. In this study, a 1327-base pair (bp) region upstream of the MrBAS of the translation start site was cloned from the genome of M. recutita. MrBAS promoter sequence analysis revealed multiple light-responsive elements, and further dark treatment reduced α-bisabolol content in flowers. The α-bisabolol content and MrBAS expression levels in various flower tissues showed a strong correlation. The 5′ deletion analysis revealed that the MrBAS promoter sequence could drive β-glucuronidase (GUS) gene expression in Nicotiana benthamiana leaves, with activity decreasing as the fragment shortened. Transgenic experiments demonstrated that the MrBAS promoter could specifically drive GUS gene expression in Arabidopsis anthers, pollen tubes, and petals. Thus, the MrBAS promoter has the potential to be a tool for directing transgene expression specifically in flower organs, offering new research avenues for cultivar development.

Recent Advancements in AAV-Vectored Immunoprophylaxis in the Nonhuman Primate Model.

Monoclonal antibodies (mAbs) are important treatment modalities for preventing and treating infectious diseases, especially for those lacking prophylactic vaccines or effective therapies. Recent advances in mAb gene cloning from naturally infected or immunized individuals has led to the development of highly potent human mAbs against a wide range of human and animal pathogens. While effective, the serum half-lives of mAbs are quite variable, with single administrations usually resulting in short-term protection, requiring repeated doses to maintain therapeutic concentrations for extended periods of time. Moreover, due to their limited time in circulation, mAb therapies are rarely given prophylactically; instead, they are generally administered therapeutically after the onset of symptoms, thus preventing mortality, but not morbidity. Adeno-associated virus (AAV) vectors have an established record of high-efficiency in vivo gene transfer in a variety of animal models and humans. When delivered to post-mitotic tissues such as skeletal muscle, brain, and heart, or to organs in which cells turn over slowly, such as the liver and lungs, AAV vector genomes assume the form of episomal concatemers that direct transgene expression, often for the lifetime of the cell. Based on these attributes, many research groups have explored AAV-vectored delivery of highly potent mAb genes as a strategy to enable long-term expression of therapeutic mAbs directly in vivo following intramuscular or intranasal administration. However, clinical trials in humans and studies in nonhuman primates (NHPs) indicate that while AAVs are a powerful and promising platform for vectored immunoprophylaxis (VIP), further optimization is needed to decrease anti-drug antibody (ADA) and anti-capsid antibody responses, ultimately leading to increased serum transgene expression levels and improved therapeutic efficacy. The following review will summarize the current landscape of AAV VIP in NHP models, with an emphasis on vector and transgene design as well as general delivery system optimization. In addition, major obstacles to AAV VIP, along with implications for clinical translation, will be discussed.

Alphaviruses in Immunotherapy and Anticancer Therapy.

Alphaviruses have been engineered as expression vectors for vaccine development and gene therapy. Due to the feature of RNA self-replication, alphaviruses can provide exceptional direct cytoplasmic expression of transgenes based on the delivery of recombinant particles, naked or nanoparticle-encapsulated RNA or plasmid-based DNA replicons. Alphavirus vectors have been utilized for the expression of various antigens targeting different types of cancers, and cytotoxic and antitumor genes. The most common alphavirus vectors are based on the Semliki Forest virus, Sindbis virus and Venezuelan equine encephalitis virus, but the oncolytic M1 alphavirus has also been used. Delivery of immunostimulatory cytokine genes has been the basis for immunotherapy demonstrating efficacy in different animal tumor models for brain, breast, cervical, colon, lung, ovarian, pancreatic, prostate and skin cancers. Typically, therapeutic effects including tumor regression, tumor eradication and complete cure as well as protection against tumor challenges have been observed. Alphavirus vectors have also been subjected to clinical evaluations. For example, therapeutic responses in all cervical cancer patients treated with an alphavirus vector expressing the human papilloma virus E6 and E7 envelope proteins have been achieved.

Advances in protoplast transfection promote efficient CRISPR/Cas9-mediated genome editing in tetraploid potato.

An efficient method of DNA-free gene-editing in potato protoplasts was developed using linearized DNA fragments, UBIQUITIN10 promoters of several plant species, kanamycin selection, and transient overexpression of the BABYBOOM transcription factor. Plant protoplasts represent a reliable experimental system for the genetic manipulation of desired traits using gene editing. Nevertheless, the selection and regeneration of mutated protoplasts are challenging and subsequent recovery of successfully edited plants is a significant bottleneck in advanced plant breeding technologies. In an effort to alleviate the obstacles related to protoplasts' transgene expression and protoplasts' regeneration, a new method was developed. In so doing, it was shown that linearized DNA could efficiently transfect potato protoplasts and that UBIQUITIN10 promoters from various plants could direct transgene expression in an effective manner. Also, the inhibitory concentration of kanamycin was standardized for transfected protoplasts, and the NEOMYCIN PHOSPHOTRANSFERASE2 (NPT2) gene could be used as a potent selection marker for the enrichment of transfected protoplasts. Furthermore, transient expression of the BABYBOOM (BBM) transcription factor promoted the regeneration of protoplast-derived calli. Together, these methods significantly increased the selection for protoplasts that displayed high transgene expression, and thereby significantly increased the rate of gene editing events in protoplast-derived calli to 95%. The method developed in this study facilitated gene-editing in tetraploid potato plants and opened the way to sophisticated genetic manipulation in polyploid organisms.

Promoter CAG is more efficient than hepatocyte‑targeting TBG for transgene expression via rAAV8 in liver tissues.

The recombinant adeno‑associated virus 8 (rAAV8) vector is a widely used tool in basic research and clinical trials. The cytomegalovirus immediate‑early enhancer/chicken β‑actin (CAG) promoter is a synthetic promoter used in adenoviral constructs with a wide spectrum and notable efficiency. The thyroxine binding globulin (TBG) promoter is a liver‑specific promoter, which directs transgene expression in hepatocytes. However, the transduction efficiency of the rAAV vector is dependent on both the administration routes and the promoter elements. In the present study, the transduction efficiency in the liver following intraperitoneal (IP) and intravenous (IV) injections of rAAV8 with the CAG, TBG669 and TBG410 promoters was compared. Enhanced green fluorescent protein (EGFP) expression was used as the biomarker to indicate efficiency. Among the three different promoters, CAG exhibited the highest efficiency from both IV and IP injections. Following IV administration, EGFP expression, induced by the CAG promoter, was 67‑fold higher compared with that in the TBG410 promoter group and 26‑fold higher compared with that in the TBG669 promoter group. EGFP protein expression was higher with IV injection compared with that for IP injection for both the CAG and TBG669 promoters (P<0.05). With the CAG promoter, EGFP protein expression was 1.5‑fold higher with the use of IV injection than with IP injection. With the TBG410 promoter, no differences were observed between the two administrations. In conclusion, these findings demonstrated that the CAG promoter was much more efficient at driving gene expression in the liver compared with that for the TBG promoters in rAAV8. In addition, IP administration produced comparable efficiency for gene delivery via the rAAV8 vector, particularly with the promoter TBG410.

Modular Lentiviral Vectors for Highly Efficient Transgene Expression in Resting Immune Cells.

Gene/cell therapies are promising strategies for the many presently incurable diseases. A key step in this process is the efficient delivery of genes and gene-editing enzymes to many cell types that may be resistant to lentiviral vector transduction. Herein we describe tuning of a lentiviral gene therapy platform to focus on genetic modifications of resting CD4+ T cells. The motivation for this was to find solutions for HIV gene therapy efforts. Through selection of the optimal viral envelope and further modification to its expression, lentiviral fusogenic delivery into resting CD4+ T cells exceeded 80%, yet Sterile Alpha Motif and HD domain 1 (SAMHD1) dependent and independent intracellular restriction factors within resting T cells then dominate delivery and integration of lentiviral cargo. Overcoming SAMHD1-imposed restrictions, only observed up to 6-fold increase in transduction, with maximal gene delivery and expression of 35%. To test if the biologically limiting steps of lentiviral delivery are reverse transcription and integration, we re-engineered lentiviral vectors to simply express biologically active mRNA to direct transgene expression in the cytoplasm. In this setting, we observed gene expression in up to 65% of resting CD4+ T cells using unconcentrated MS2 lentivirus-like particles (MS2-LVLPs). Taken together, our findings support a gene therapy platform that could be readily used in resting T cell gene editing.

Optimising the new ucoe models as higher direct transgene expression profile for effective gene therapy applications

Background & Aim: Our new chromatin opening models which free from potential mutation sites that reduce the size of the current UCOE models used in gene therapy and recombinant protein biotechnology studies, tested on various cell groups, including mouse stem cell groups. As a result, it has been demonstrated in terms of replacing existing UCOE models that these new UCOE candidates we have developed are more efficient than the previous ones and that they are also a safer profile model for clinical gene therapy studies since they are free from additional cassette areas. To see the potentiality of our new generation UCOE designs in gene therapy studies, it was tested whether the universal chromatin opening abilities will be retained stable of activation on human induced pluripotent stem cells by differentiating them into different tissue cell types as done before on mouse embryonic stem cells. Methods, Results & Conclusion: After transfection of induced human pluripotent stem cells with Lentiviral vectors carrying the UCOE [Figure presented] [Figure presented] candidates, the activation of UCOE elements which carry a reporter gene, were measured by flowytometry, immunofluorescence staining and DNA methylation analyses for two months without differentiation and also after tissue type differentiation into nerve, hepatocyte and cardiomycet type cells. In the light of the obtained results, it has been observed that the new UCOE designs (1.2kb and 1.7kb UCOE chromatin elements) that we developed, have maintained their expression levels stably on human iPS cells before and after differentiation into three different tissue type cells. And additionally, they also showed their potential on monoclonal antibody production with CHO cells as producing mg and gr level of recombinant antibodies into two months of period in another parallel study. In addition, our new 0.5kb design (0.5kb UCOE), produced from another CpG density region of HNRPA2B1 gene, has been observed to display a stable expression level compared to our other designs and even a more stable profile. The new 0.5kb, 1.2kb 1.7kb (without expression enhancing cassette regions, thus eliminating the potential mutation problem) with a length of 10, 6 and 3 times shorter than the current standard A2UCOE models showed to be more advantageous for gene therapy and recombinant protein production studies as well. Recently, we have been using these new models now on producing the two of the target antigens of COVID-19 to provide a potential recombinant vaccine candidate.

Direct transgene expression and mAb production by using new model UCOEs on iPS and CHO cells

Background & Aim It has recently been shown that efficient and stable expression of transgenes from the A2UCOE is at least in part due to its resistance to DNA methylation- mediated silencing. Analysis of a deletion series from the CBX3 end of a fully functional 2.5kb A2UCOE where expression of an eGFP reporter gene is directly driven off the HNRPA2B1 promoter, revealed a 1.2kb and 1.7kb truncation that retained full UCOE activity following transduction of P19 and F9 cells in both undifferentiated and differentiated states. These 1.2-1.7 and a further deletion 0.5kb A2UCOEs, were also able to retain stable expression in murine embryonic stem cells and now in human iPS cells during differentiation into embryoid bodies and three different tissue specific cell types. In addition, from a practical perspective our finding that the 0.5-1.2-1.7A2UCOEs retain the same stability of expression as the larger 2.5kb A2UCOE model suggests that it can effectively replace the latter within therapeutic LV constructs allowing a greater capacity and also more safety for the gene of interest by cutting off the enhancer elements which carries a potential mutation risk. A further project now on human mAb production with recombinant CHO cells by using our new UCOE models has been started and the early results show that they are more productive than the control A2UCOEs and able to produce human recombinant mAbs in mg to g level even under two months of cultivation. The results already encouraging to provide a new better UCOE model for recombinant protein production in mammalian cells. Methods, Results & Conclusion Firstly, the new UCOE models were cloned into lentiviral vectors and then the vectors were produced by 293T cells. Then, human iPS cells were transfected by the vectors without differentiation. The expression of the UCOE models were observed over 42 days by measuring eGfP+ cell levels in flow cytometry every week. The experiments then were followed by differentiation of transduced iPS cells into three different tissue spesific cell types (pre and neuronal, hepatocyte and cardiomyocyte cell types). Secondly, the aimed mAb DNA sequences were produced by PCR, and clonned into our UCOE vectors and then they were transduced into the CHO group sub-cell clones which the recombinant protein production will be done and the mAbs will then be isolated from the cell culture and analysed by correction methods. After the correction analyses, the production of aimed IgG1 ve IgE/k group mAbs were produced in bioreactor in gram level.

High-level protein production in erythroid cells derived from in vivo transduced hematopoietic stem cells

AbstractWe developed an in vivo hematopoietic stem cell (HSC) transduction approach that involves HSC mobilization from the bone marrow into the peripheral bloodstream and the IV injection of an integrating, helper-dependent adenovirus (HDAd5/35++) vector system. HDAd5/35++ vectors target human CD46, a receptor that is abundantly expressed on primitive HSCs. Transgene integration is achieved by a hyperactive Sleeping Beauty transposase (SB100x) and transgene marking in peripheral blood cells can be increased by in vivo selection. Here we directed transgene expression to HSC-derived erythroid cells using β-globin regulatory elements. We hypothesized that the abundance and systemic distribution of erythroid cells can be harnessed for high-level production of therapeutic proteins. We first demonstrated that our approach allowed for sustained, erythroid-lineage specific GFP expression and accumulation of GFP protein in erythrocytes. Furthermore, after in vivo HSC transduction/selection in hCD46-transgenic mice, we demonstrated stable supraphysiological plasma concentrations of a bioengineered human factor VIII, termed ET3. High-level ET3 production in erythroid cells did not affect erythropoiesis. A phenotypic correction of bleeding was observed after in vivo HSC transduction of hCD46+/+/F8−/− hemophilia A mice despite high plasma anti-ET3 antibody titers. This suggests that ET3 levels were high enough to provide sufficient noninhibited ET3 systemically and/or locally (in blood clots) to control bleeding. In addition to its relevance for hemophilia A gene therapy, our approach has implications for the therapy of other inherited or acquired diseases that require high levels of therapeutic proteins in the blood circulation.

Enhanced vascular activity of a new chimeric promoter containing the full CaMV 35S promoter and the plant XYLOGEN PROTEIN 1 promoter.

To develop a new strategy that controls vascular pathogen infections in economic crops, we examined a possible enhancer of the vascular activity of XYLOGEN PROTEIN 1 promoter (Px). This protein is specifically expressed in the vascular tissues of Arabidopsis thaliana and plays an important role in xylem development. Although Px is predicted as vascular-specific, its activity is hard to detect and highly susceptible to plant and environmental conditions. The cauliflower mosaic virus 35S promoter (35S) is highly active in directing transgene expression. To test if 35S could enhance Px activity, while vascular specificity of the promoter is retained, we examined the expression of the uidA reporter gene, which encodes β-glucuronidase (GUS), under the control of a chimeric promoter (35S-Px) or Px by generating 35S-Px-GUS and Px-GUS constructs, which were transformed into tobacco seedlings. Both 35S-Px and Px regulated gene expression in vascular tissues. However, GUS expression driven by 35S-Px was not detected in 30- and 60-day-old plants. Quantitative real-time PCR analysis showed that GUS gene expression regulated by 35S-Px was 6.2-14.9-fold higher in vascular tissues than in leaves. Histochemical GUS staining demonstrated that 35S-Px was strongly active in the xylem and phloem. Thus, fusion of 35S and Px might considerably enhance the strength of Px and increase its vascular specificity. In addition to confirming that 35S enhances the activity of a low-level tissue-specific promoter, these findings provide information for further improving the activity of such promoters, which might be useful for engineering new types of resistant genes against vascular infections.

A profilin gene promoter from switchgrass (Panicum virgatum L.) directs strong and specific transgene expression to vascular bundles in rice

A switchgrass vascular tissue-specific promoter (PvPfn2) and its 5'-end serial deletions drive high levels of vascular bundle transgene expression in transgenic rice. Constitutive promoters are widely used for crop genetic engineering, which can result in multiple off-target effects, including suboptimal growth and epigenetic gene silencing. These problems can be potentially avoided using tissue-specific promoters for targeted transgene expression. One particularly urgent need for targeted cell wall modification in bioenergy crops, such as switchgrass (Panicum virgatum L.), is the development of vasculature-active promoters to express cell wall-affective genes only in the specific tissues, i.e., xylem and phloem. From a switchgrass expression atlas we identified promoter sequence upstream of a vasculature-specific switchgrass profilin gene (PvPfn2), especially in roots, nodes and inflorescences. When the putative full-length (1715bp) and 5'-end serial deletions of the PvPfn2 promoter (shortest was 413bp) were used to drive the GUS reporter expression in stably transformed rice (Oryza sativa L.), strong vasculature-specificity was observed in various tissues including leaves, leaf sheaths, stems, and flowers. The promoters were active in both phloem and xylem. It is interesting to note that the promoter was active in many more tissues in the heterologous rice system than in switchgrass. Surprisingly, all four 5'-end promoter deletions, including the shortest fragment, had the same expression patterns as the full-length promoter and with no attenuation in GUS expression in rice. These results indicated that the PvPfn2 promoter variants are new tools to direct transgene expression specifically to vascular tissues in monocots. Of special interest is the very compact version of the promoter, which could be of use for vasculature-specific genetic engineering in monocots.

Evaluating Tissue-Specific Recombination in a Pdgfrα-CreERT2 Transgenic Mouse Line.

In the central nervous system (CNS) platelet derived growth factor receptor alpha (PDGFRα) is expressed exclusively by oligodendrocyte progenitor cells (OPCs), making the Pdgfrα promoter an ideal tool for directing transgene expression in this cell type. Two Pdgfrα-CreERT2 mouse lines have been generated for this purpose which, when crossed with cre-sensitive reporter mice, allow the temporally restricted labelling of OPCs for lineage-tracing studies. These mice have also been used to achieve the deletion of CNS-specific genes from OPCs. However the ability of Pdgfrα-CreERT2 mice to induce cre-mediated recombination in PDGFRα+ cell populations located outside of the CNS has not been examined. Herein we quantify the proportion of PDGFRα+ cells that become YFP-labelled following Tamoxifen administration to adult Pdgfrα-CreERT2::Rosa26-YFP transgenic mice. We report that the vast majority (>90%) of PDGFRα+ OPCs in the CNS, and a significant proportion of PDGFRα+ stromal cells within the bone marrow (~38%) undergo recombination and become YFP-labelled. However, only a small proportion of the PDGFRα+ cell populations found in the sciatic nerve, adrenal gland, pituitary gland, heart, gastrocnemius muscle, kidney, lung, liver or intestine become YFP-labelled. These data suggest that Pdgfrα-CreERT2 transgenic mice can be used to achieve robust recombination in OPCs, while having a minimal effect on most PDGFRα+ cell populations outside of the CNS.

Hematopoietic Stem Cells Are the Major Source of Multilineage Hematopoiesis in Adult Animals

Hematopoietic stem cells (HSCs) sustain long-term reconstitution of hematopoiesis in transplantation recipients, yet their role in the endogenous steady-state hematopoiesis remains unclear. In particular, recent studies suggested that HSCs provide a relatively minor contribution to immune cell development in adults. We directed transgene expression in a fraction of HSCs that maintained reconstituting activity during serial transplantations. Inducible genetic labeling showed that transgene-expressing HSCs gave rise to other phenotypic HSCs, confirming their top position in the differentiation hierarchy. The labeled HSCs rapidly contributed to committed progenitors of all lineages and to mature myeloid cells and lymphocytes, but not to B-1a cells or tissue macrophages. Importantly, labeled HSCs gave rise to more than two-thirds of all myeloid cells and platelets in adult mice, and this contribution could be accelerated by an induced interferon response. Thus, classically defined HSCs maintain immune cell development in the steady state and during systemic cytokine responses.

Locally Targeted Cardiac Gene Delivery by AAV Microbubble Destruction in a Large Animal Model.

Cardiac gene therapy is a promising approach for treating heart diseases. Although clinical studies are ongoing, effective and targeted transgene delivery is still a major obstacle. We sought to improve and direct transgene expression in myocardium by ultrasound-targeted microbubble destruction (UTMD). In pigs, adeno-associated virus-derived (AAV) vectors harboring the luciferase reporter gene were delivered via retroinfusion into the anterior interventricular coronary vein (AIV). AAV vectors were either loaded to lipid microbubbles before injection or injected unmodified. Upon injection of AAV/microbubble solution, UTMD was performed. After 4 weeks, reporter gene expression levels in the anterior wall (target area), in the posterior wall (control area), and in noncardiac organs were analyzed. Retroinfusion of AAV-luciferase vectors loaded to lipid microbubbles led to a significant increase in transgene expression, with an increase in UTMD targeted areas of the anterior wall. Moreover, off-target expression was reduced in comparison to control animals, receiving AAV-luciferase without microbubbles. Besides an increase in overall target area transgene expression, UTMD alters the spatial expression of the luciferase transgene, focusing expression to ultrasound-targeted left ventricular wall. These data suggest UTMD as a promising approach for directing AAV to specific cardiac segments.

Sacasg and sacalg: new GFP-labelled camel skin and lung fibroblast cell lines

Camelids skin is claimed to respond differently under different physiological and pathological condition, however, there is no in vitro model with a reporter to investigate this claim. So generating camel fibroblast cell lines that expresses green fluorescent protein (GFP) would be important as a tool to monitor camel cell growth, migration and other processes. Hence, in this investigation, we created a GFP expressing fibroblast cell lines derived from a primary skin and lung fibroblast cell lines of the Arabian camel with a stable expression of GFP which was transfected using the pCAG-EGFP plasmid with CMV enhancer via Lipofectamine 2000 reagent. Stably transfected clones were selected by puromycin screening. The results revealed that camel fibroblasts can be efficiently transduced in vitro using pCAG-CMV-based vectors and that these vectors direct long-term transgene expression without evident toxicity, pathogenesis or alteration of native fibroblast morphology. Immunofluorescence staining showed no differences in the expression of fibroblast biomarkers between the transfected and non-transfected cell lines. The viability of thawed cells remained above 85% after cryopreservation in liquid nitrogen. The gene expression of the fibroblast markers was not different in the transfected cell lines. Taken together, we have established and fully characterised GFP expressing-fibroblast cell lines of Arabian camel. Based on our assays, we conclude that transfection of GFP into the Arabian camel skin and lung fibroblasts did not change their observed properties. The GFP-labelled cell lines may represent a new tool for convenient monitoring of live primary camel fibroblasts.

Soybean actin, heat shock protein, and ribosomal protein promoters direct tissue-specific transgene expression in transgenic soybean

Soybean (Glycine max) promoters from an actin gene (GmActin), a ribosomal protein S11 gene (GmRpS11) and a heat shock protein 90 gene (GmHsp90), identified as being active during early induction of soybean somatic embryogenesis, were cloned, fused to the green fluorescence protein (gfp) gene and reintroduced into soybean for evaluation. All promoters displayed development- and tissue-specific regulation based on the expression of GFP in transgenic soybean. In seedlings, these three promoters gave rise to strong GFP expression in young roots, with very high levels of expression in the vascular tissues and root primordia. In older plants, promoter activity was observed in the vascular cambium of petioles, leaf midribs, and in the mesophyll of expanding leaves, but at reduced levels. The GmActin promoter gave very high GFP expression in developing and mature seeds. All three promoters displayed high activity following induction of somatic embryogenesis from immature soybean cotyledons. Comparison of promoter-regulated GFP transgene expression with transcriptome analysis using RNA-Seq revealed agreement in expression intensity in roots, leaves, and flowers but exceptions for expression in young developing seeds. Evaluation of specific promoters in transgenic plants is needed for proper elucidation of promoter-regulated transgene activity.

The pineapple AcMADS1 promoter confers high level expression in tomato and Arabidopsis flowering and fruiting tissues, but AcMADS1 does not complement the tomato LeMADS-RIN (rin) mutant.

A previous EST study identified a MADS box transcription factor coding sequence, AcMADS1, that is strongly induced during non-climacteric pineapple fruit ripening. Phylogenetic analyses place the AcMADS1 protein in the same superclade as LeMADS-RIN, a master regulator of fruit ripening upstream of ethylene in climacteric tomato. LeMADS-RIN has been proposed to be a global ripening regulator shared among climacteric and non-climacteric species, although few functional homologs of LeMADS-RIN have been identified in non-climacteric species. AcMADS1 shares 67 % protein sequence similarity and a similar expression pattern in ripening fruits as LeMADS-RIN. However, in this study AcMADS1 was not able to complement the tomato rin mutant phenotype, indicating AcMADS1 may not be a functionally conserved homolog of LeMADS-RIN or has sufficiently diverged to be unable to act in the context of the tomato network of interacting proteins. The AcMADS1 promoter directed strong expression of the GUS reporter gene to fruits and developing floral organs in tomato and Arabidopsis thaliana, suggesting AcMADS1 may play a role in flower development as well as fruitlet ripening. The AcMADS1 promoter provides a useful molecular tool for directing transgene expression, particularly where up-regulation in developing flowers and fruits is desirable.

The Expanding Genetic Toolkit for Exploring Mechanisms of General Anesthesia

Serious scientific efforts aimed at understanding how general anesthetics produce their powerful effects began soon after the historic demonstration of ether anesthesia on October 16, 1846 in Boston, Massachusetts. Progress was slow for most of the intervening 166 years, but has dramatically advanced during the last three decades, aided by new ideas and the revolutionary techniques of molecular biology. One result of this progress is the report by Zhou et al1 in this month's Anesthesiology, describing studies on ketamine in conditional knockout animals. Here, I briefly sketch how genetic techniques have spurred our understanding of general anesthetic mechanisms.

A Novel Moderate Constitutive Promoter Derived from Poplar (Populus tomentosa Carrière).

A novel sequence that functions as a promoter element for moderate constitutive expression of transgenes, designated as the PtMCP promoter, was isolated from the woody perennial Populus tomentosa. The PtMCP promoter was fused to the GUS reporter gene to characterize its expression pattern in different species. In stable Arabidopsis transformants, transcripts of the GUS reporter gene could be detected by RT-PCR in the root, stem, leaf, flower and silique. Further histochemical and fluorometric GUS activity assays demonstrated that the promoter could direct transgene expression in all tissues and organs, including roots, stems, rosette leaves, cauline leaves and flowers of seedlings and maturing plants. Its constitutive expression pattern was similar to that of the CaMV35S promoter, but the level of GUS activity was significantly lower than in CaMV35S promoter::GUS plants. We also characterized the promoter through transient expression in transgenic tobacco and observed similar expression patterns. Histochemical GUS staining and quantitative analysis detected GUS activity in all tissues and organs of tobacco, including roots, stems, leaves, flower buds and flowers, but GUS activity in PtMCP promoter::GUS plants was significantly lower than in CaMV35S promoter::GUS plants. Our results suggested that the PtMCP promoter from poplar is a constitutive promoter with moderate activity and that its function is presumably conserved in different species. Therefore, the PtMCP promoter may provide a practical choice to direct moderate level constitutive expression of transgenes and could be a valuable new tool in plant genetic engineering.

Identification and Cytoprotective Function of a Novel Nestin Isoform, Nes-S, in Dorsal Root Ganglia Neurons

In this study, the first nestin isoform, Nes-S, was identified in neurons of dorsal root ganglia (DRG) of adult rats. Nes-S cannot form filaments by itself in cytoplasmic intermediate filament-free SW13 cells. Instead, it co-assembles into filaments with vimentin when transfected into vimentin(+) SW13 cells, and with peripherin and neurofilament proteins when transfected into N2a cells. In primary DRG neurons, endogenous Nes-S co-assembles with peripherin and neurofilament proteins. The expression of Nes-S first appears in DRG at postnatal day 5 and persists to adulthood. Among the adult tissues we examined, the expression of Nes-S is restricted to the sensory and motor neurons. Finally, exogenous Nes-S enhances viability when transfected into N2a cells, and knockdown of endogenous Nes-S impairs the survival of DRG neurons in primary cultures. Taken together, Nes-S is a new neuronal intermediate filament protein that exerts a cytoprotective function in mature sensory and motor neurons.