Oct4‐induced oligodendrocyte progenitor cells enhance functional recovery in spinal cord injury model

The generation of patient‐specific oligodendrocyte progenitor cells (OPCs) holds great potential as an expandable cell source for cell replacement therapy as well as drug screening in spinal cord injury or demyelinating diseases. Here, we demonstrate that induced OPCs (iOPCs) can be directly derived from adult mouse fibroblasts by Oct4‐mediated direct reprogramming, using anchorage‐independent growth to ensure high purity. Homogeneous iOPCs exhibit typical small‐bipolar morphology, maintain their self‐renewal capacity and OPC marker expression for more than 31 passages, share high similarity in the global gene expression profile to wild‐type OPCs, and give rise to mature oligodendrocytes and astrocytes in vitro and in vivo. Notably, transplanted iOPCs contribute to functional recovery in a spinal cord injury (SCI) model without tumor formation. This study provides a simple strategy to generate functional self‐renewing iOPCs and yields insights for the in‐depth study of demyelination and regenerative medicine.     

Overexpression and purification of full-length acidic fibroblast growth factor inE.coli

Summary A full-length aFGF cDNA was cloned and inserted into a T7 expression vector for heterologous expression inE. coli. For high level production of aFGF, an optimal condition for overexpression (incubation at 42 °C for 2 hours) and a simple purification procedure employingin vitro refolding and an affinity chromatography was established. The production yield was 40–50 μg/mg of inclusion body preparation and purified aFGF showed mitogenic activity.     

High level expression and purification of active recombinant human interleukin-8 in Pichia pastoris

Human interleukin-8 (hIL-8) is a member of interleukin family which functions as a chemotactic factor as well as an angiogenesis mediator. Previously, a study reported that hIL-8 could be purified from inclusion bodies using a prokaryotic expression system, however, the required re-naturation step limits the recovery of fully active protein. In this study, soluble recombinant hIL-8 was expressed as a secreted protein at high level in Pichia pastoris under the control of AOX1 (alcohol oxidase 1) promoter. A simple purification strategy was established to recover rhIL-8 from the fermentation supernatant. The process includes precipitation with 80% saturation ammonium sulfate and CM Sepharose ion exchange chromatography, yielding 30 mg/L purified rhIL-8 at over 95% purity. The obtained rhIL-8 displays high specific activity, stimulating the migration of mouse neutrophils at concentrations as low as 0.25 ng/mL. Our results demonstrate that P. pastoris expression system is an efficient tool for large-scale manufacture of active recombinant hIL-8 for various applications.     

Outlook in the application of Chlamydomonas reinhardtii chloroplast as a platform for recombinant protein production

Microalgae, also called microphytes, are a vast group of microscopic photosynthetic organisms living in aquatic ecosystems. Microalgae have attracted the attention of biotechnology industry as a platform for extracting natural products with high commercial value. During last decades, microalgae have been also used as cost-effective and easily scalable platform for the production of recombinant proteins with medical and industrial applications. Most progress in this field has been made with Chlamydomonas reinhardtii as a model organism mainly because of its simple life cycle, well-established genetics and ease of cultivation. However, due to the scarcity of existing infrastructure for commercial production and processing together with relatively low product yields, no recombinant products from C. reinhardtii have gained approval for commercial production and most of them are still in research and development. In this review, we focus on the chloroplast of C. reinhardtii as an algal recombinant expression platform and compare its advantages and disadvantages to other currently used expression systems. We then discuss the strategies for engineering the chloroplast of C. reinhardtii to produce recombinant cells and present a comprehensive overview of works that have used this platform for the expression of high-value products.     

Co-integration, co-expression and inheritance of unlinked minimal transgene expression cassettes in an apomictic turf and forage grass (Paspalum notatum Flugge)

Bahiagrass (Paspalum notatum Flugge) is an important turf and forage grass in the southeastern United States and other subtropical regions. Biolistic co-transfer of two unlinked, minimal, linear transgene expression cassettes (MCs) into the apomictic bahiagrass cv. Argentine was carried out to evaluate co-integration, quantify co-expression and analyze inheritance to apomictic seed progeny. Gold projectiles were coated with minimal unlinked nptII and bar expression cassettes in a 1:2 molar ratio. Complexity of transgene loci correlated with the amount of DNA used during gene transfer. Transgenic plants displayed a simple nptII integration pattern with 1–4 hybridization signals compared to the non-selected bar gene with 2 to more than 5 hybridization signals per transgenic line. Co-expression of unlinked nptII and bar genes occurred in 19 of the 20 co-transformed lines (95% co-expression frequency). Protein quantification revealed that several lines with complex integration patterns displayed a higher transgene expression than lines with simple transgene integration patterns. Several transgenic lines displayed hybridization signals indicative of concatemerization. Concatemers were confirmed following PCR amplification and sequence analysis of transgene loci. The obligate apomictic bahiagrass cv. Argentine produced uniform seed progeny without segregation of simple or complex transgene loci. NPTII- and PAT-ELISA, as well as herbicide application, confirmed stable expression of the nptII and bar gene at levels similar to the primary transformants. These results demonstrate that biolistic transfer of MCs support stable and high level co-expression of transgenes in bahiagrass.     

Heterologous expression of L. major proteins in S. cerevisiae: a test of solubility, purity, and gene recoding

High level expression of many eukaryotic proteins for structural analysis is likely to require a eukaryotic host since many proteins are either insoluble or lack essential post-translational modifications when expressed in E. coli. The well-studied eukaryote Saccharomyces cerevisiae possesses several attributes of a good expression host: it is simple and inexpensive to culture, has proven genetic tractability, and has excellent recombinant DNA tools. We demonstrate here that this yeast exhibits three additional characteristics that are desirable in a eukaryotic expression host. First, expression in yeast significantly improves the solubility of proteins that are expressed but insoluble in E. coli. The expression and solubility of 83 Leishmania major ORFs were compared in S. cerevisiae and in E. coli, with the result that 42 of the 64 ORFs with good expression and poor solubility in E. coli are highly soluble in S. cerevisiae. Second, the yield and purity of heterologous proteins expressed in yeast is sufficient for structural analysis, as demonstrated with both small scale purifications of 21 highly expressed proteins and large scale purifications of 2 proteins, which yield highly homogeneous preparations. Third, protein expression can be improved by altering codon usage, based on the observation that a codon-optimized construct of one ORF yields three-fold more protein. Thus, these results provide direct verification that high level expression and purification of heterologous proteins in S. cerevisiae is feasible and likely to improve expression of proteins whose solubility in E. coli is poor.     

Transformation of Plants with Multiple Cassettes Generates Simple Transgene Integration Patterns and High Expression Levels

We transformed rice (Oryza sativa L.) simultaneously with five minimal cassettes, each containing a promoter, coding region and polyadenylation site but no vector backbone. We found that multi-transgene cotransformation was achieved with high efficiency using multiple cassettes, with all transgenic plants we generated containing at least two transgenes and 16% containing all five. About 75% of the plants had simple transgene integration patterns with a predominance of single-copy insertions. The expression levels for all transgenes, and the overall coexpression frequencies, were much higher than previously reported in whole plasmid transformants. Four of five lines analyzed for transgene expression stability in subsequent generations showed stable and high expression levels over generations. A simple model is proposed, which accounts for differences in the molecular make-up and the expression profile of transgenic plants generated using whole plasmid or minimal cassettes. We conclude that gene transfer using minimal cassettes is an efficient and rapid method for the production of transgenic plants containing and stably expressing several different transgenes. Our results facilitate effective manipulation of multi-gene pathways in plants in a single transformation step.     

Thermostable lichenase as a translational reporter

Hybrid genes containing the reporter gene for thermostable lichenase and model genes recA, recA1, cry3a, cry3aM, and ssp1 were constructed. The expression of these genes was studied in prokaryotic and eukaryotic cells. The presence of lichenase in the hybrid proteins was shown to facilitate analysis of the hybrid protein expression in transgenic organisms. Owing to high relative activity and thermostability of lichenase, the activity of this enzyme can be measured by simple, rapid and sensitive qualitative and quantitative methods that do not require costly equipment and reagents. Using the zymograms method, molecular masses of the lichenase-containing hybrid proteins can be precisely estimated. This method is proposed instead of Western blotting using lichenase as a translational reporter. Our results showed that the use of thermostable lichenase as a translational reporter yields the data that are problematic to obtain using traditional methods of gene expression analysis, which is of importance for fundamental and applied research.Translated from Genetika, Vol. 41, No. 1, 2005, pp. 30–39.Original Russian Text Copyright © 2005 by Komakhin, Abdeeva, Salehi Dzhuzani, Goldenkova, Zhuchenko.     

A large‐scale gene discovery for the red palm weevil Rhynchophorus ferrugineus (Coleoptera: Curculionidae)

The red palm weevil (RPW; Rhynchophorus ferrugineus) is a devastating pest of palms, prevalent in the Middle East as well as many other regions of the world. Here, we report a large‐scale de novo complementary DNA (cDNA) sequencing effort that acquired ～5 million reads and assembled them into 26 765 contigs from 12 libraries made from samples of different RPW developmental stages based on the Roche/454 GS FLX platform. We annotated these contigs based on the publically available known insect genes and the Tribolium castaneum genome assembly. We find that over 80% of coding sequences (CDS) from the RPW contigs have high‐identity homologs to known proteins with complete CDS. Gene expression analysis shows that the pupa and larval stages have the highest and lowest expression levels, respectively. In addition, we also identified more than 60 000 single nucleotide polymorphisms and 1 200 simple sequence repeat markers. This study provides the first large‐scale cDNA dataset for RPW, a much‐needed resource for future molecular studies.     

High intensity and blue light regulated expression of chimeric chalcone synthase genes in transgenic Arabidopsis thaliana plants

Summary To establish a genetic system for dissection of light-mediated signal transduction in plants, we analyzed the light wavelengths and promoter sequences responsible for the light-induced expression of the Arabidopsis thaliana chalcone synthase (CHS) promoter fused to the -glucuronidase (GUS) marker gene. Transgenic A. thaliana lines carrying 1975, 523, 186, and 17 by of the CHS promoter fused to the GUS gene were generated, and the expression of these chimeric genes was monitored in response to high intensity light in mature plants and to different wavelengths of light in seedlings. Fusion constructs containing 1975 and 523 by of CHS promoter sequence behaved identically to the endogenous CHS gene under all conditions. Expression of these constructs was induced specifically in response to high intensity white light and blue light. The response to blue light was seen in the presence of the P_fr form of phytochrome. Fusion constructs containing 186 by of promoter sequence showed reduced basal levels of expression and only weak stimulation by blue light but were induced significantly by high intensity white light. These analyses showed that the expression of the A. thaliana CHS gene is responsive to a specific blue light receptor and that sequences between — 523 and — 186 by are required for optimal basal and blue light-induced expression of this gene. The experiments lay the foundation for a simple genetic screen for light response mutants.     

The selection of parents for synthetic varieties of outbreeding diploid crops

Summary As a criterion for the selection from a population of individuals with a high potential as parents of synthetic varieties, the general varietal ability of an individual is defined as the mean expression of all possible synthetics of a given size(s) having this plant as a common parent. Using known expressions for the prediction of the performance of advanced generations of diploid synthetic varieties, general varietal ability is expressed in terms of the F ₁ and I ₁ progenies of the plants under test, and is found to be a simple function of the polycross (g.c.a.) and inbred progeny means, where the contribution of the inbred progeny varies according to n and s. The implications and use of such a progeny test in the breeding of out-pollinating crops is discussed.     

Flexible manipulation of Omb levels in the endogenous expression region of Drosophila wing by combinational overexpression and suppression strategy

Manipulating an exogenous or endogenous gene of interest at a defined level is critical for a wide variety of experiments.The Gal4/UAS system has been widely used to direct gene expression for studying complex genetic and biological problems in Drosophila melanogaster and other model organisms.Driven by a given tissue-specific Gal4,expressing UAS-transgene or UAS-RNAi(RNA interference)could be used to up-or down-regulate target gene expression,respectively.However,the efficiency of the Gal4/UAS system is roughly predefined by properties of transposon vector constructs and the insertion site in the transgenic stock.Here,we describe a simple way to modulate optomotor blind(omb)expression levels in its endogenous expression region of the wing disc.We co-expressed UAS-omb and UAS-omb-RNAi together under the control of dpp-Gal4 driver which is expressed in the omb expression region of the wing pouch.The repression effect is more sensitive to temperature than that of overexpression.At low temperature,overexpression plays a dominant role but the efficiency is attenuated by UAS-omb-RNAi.In contrast,at high temperature RNAi predominates in gene expression regulation.By this strategy,we could manipulate omb expression levels at a moderate level.It allows us to manipulate omb expression levels in the same tissue between overexpression and repression at different stages by temperature control.     

A simple method forIn Situ hybridization to RNA in guard cells ofVicia Faba L.: The expression of aquaporins in guard cells

Because the epidermis ofV. faba L. leaves easily can be peeled into strips of one cell layer, we developed a simple method ofin situ hybridization using epidermal peels as a substitute for paraffin, resin and cryosections. Our method sufficiently detected the expression of broad bean aquaporin 1 in guard cells. RT-PCR revealed higher expression of aquaporins (AQPs) in guard cells compared to other leaf cell types; this indicates the importance of AQP for bulk water flow across guard cell membranes and, therefore, for stomatal movements.     

Nicotiana chloroplast genome

Summary RuBPCase, the enzyme responsible for carboxylation and oxidation of RuBP in a wide variety of photosynthetic organisms, is the major protein found in the chloroplast. Here we present the first evidence for direct expression in E. coli and B. subtilis of tobacco and Chlamydomonas ct-DNA sequences coding for the LS of RuBPCase as demonstrated by a simple in situ immunoassay.     

UsingGCN4as a Reporter of eIF2α Phosphorylation and Translational Regulation in Yeast

Molecular genetic analyses in yeast are a powerful method to study gene regulation. Conservation of the mechanism and regulation of protein synthesis between yeast and mammalian cells makes yeast a good model system for the analysis of translation. One of the most common mechanisms of translational regulation in mammalian cells is the phosphorylation of serine-51 on the α subunit of the translation initiation factor eIF2, which causes an inhibition of general translation. In contrast, in the yeastSaccharomyces cerevisiaephosphorylation of eIF2α on serine-51 by theGCN2protein kinase mediates the translational induction ofGCN4expression. The unique structure of theGCN4mRNA makesGCN4expression especially sensitive to eIF2α phosphorylation, and the simple microbiological tests developed in yeast to analyzeGCN4expression serve as good reporters of eIF2α phosphorylation. It is relatively simple to express heterologous proteins in yeast, and it has been shown that the mammalian eIF2α kinases will functionally substitute forGCN2.Structure–function analyses of translation factors or translational regulators can also be performed by assaying for effects on general andGCN4-specific translation. Three tests can be used to study eIF2α phosphorylation and/or translational activity in yeast. First, general translation can be monitored by simple growth tests, whileGCN4expression can be analyzed using sensitive replica-plating tests. Second,GCN4translation can be quantitated by measuring expression fromGCN4–lacZreporter constructs. Finally, isoelectric focusing gels can be used to directly monitorin vivophosphorylation of eIF2α in yeast.     

In VitroMeasurement of β-Lactamase-Catalyzed Ampicillin Hydrolysis by RecombinantEscherichia coliExtracts Using Quantitative High-Performance Liquid Chromatography

We report a rapid and simple protocol for measuring the β-lactamase activity from recombinantEscherichia colicells transformed with any of the common plasmid vectors that provide ampicillin resistance through constitutive expression and periplasmic localization of the β-lactamase TEM-1. The hydrolytic enzyme was extracted from theE. coliperiplasm and the β-lactamase activity determined by measuring conversion of ampicillin to aminobenzyl-penicilloic acid using quantitative high-performance liquid chromatography. Under saturating conditions thein vitroassay was linear as a function of both incubation time and enzyme concentration. Application of this assay to investigate TEM-1 expression, from two different protein expression vector systems, demonstrated the potential importance of this assay in studies of recombinant protein expression and translocation.     

Approaches for refining heterologous protein production in filamentous fungi

Fungi combine the advantages of a microbial system such as a simple fermentability with the capability of secreting proteins that are modified according to a general eukaryotic scheme. Filamentous fungi such as Aspergillus niger efficiently secrete genuine proteins but the secretion of recombinant proteins turned out be a difficult task. Aspergillus niger is an attractive organism because of its high secretion capacity and is frequently used as a model organism. Whereas high production yields can be obtained when homologous proteins are expressed, much lower amounts are obtained with the production of heterologous proteins. To fully exploit the potential of filamentous fungi, understanding of the molecular genetics, their physiology, and the glycosylation metabolism has to be investigated and clarified in more detail. This review summarizes recent developments in heterologous protein production by filamentous fungi and also generalizes the possibilities of improving the protein production by various genetic and bioprocessing approaches, thereby easing recognition of filamentous fungi as a relevant and reliable expression platform.