The EF‐1α promoter maintains high‐level transgene expression from episomal vectors in transfected CHO‐K1 cells

In our previous study, we demonstrated that episomal vectors based on the characteristic sequence of matrix attachment regions (MARs) and containing the cytomegalovirus (CMV) promoter allow transgenes to be maintained episomally in Chinese hamster ovary (CHO) cells. However, the transgene expression was unstable and the number of copies was low. In this study, we focused on enhancers, various promoters and promoter variants that could improve the transgene expression stability, expression magnitude (level) and the copy number of a MAR‐based episomal vector in CHO‐K1 cells. In comparison with the CMV promoter, the eukaryotic translation elongation factor 1 α (EF‐1α, gene symbol EEF1A1) promoter increased the transfection efficiency, the transgene expression, the proportion of expression‐positive clones and the copy number of the episomal vector in long‐term culture. By contrast, no significant positive effects were observed with an enhancer, CMV promoter variants or CAG promoter in the episomal vector in long‐term culture. Moreover, the high‐expression clones harbouring the EF‐1α promoter tended to be more stable in long‐term culture, even in the absence of selection pressure. According to these findings, we concluded that the EF‐1α promoter is a potent regulatory sequence for episomal vectors because it maintains high transgene expression, transgene stability and copy number. These results provide valuable information on improvement of transgene stability and the copy number of episomal vectors.     

The Amount of BCL6 in B Cells Shortly after Antigen Engagement Determines Their Representation in Subsequent Germinal Centers

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Induction of Expression Instability of the nptII Gene in Transgenic Tobacco Plants

A comparative analysis of the neomycin phosphotransferase (nptII) gene expression was performed in two groups of transformed tobacco plants, one of which included plants with direct and inverted tandem uidA gene repeats in the T-DNA insertion. This insertion of inverted repeats was shown to reduce the level of stable nptII gene expression to 20%, as compared with 65% in the control transformants. The level of unstable expression of this gene substantially increased (up to 71.4% vs. 5.5% in the control group) when homologous sequences were brought together with direct tandem repeats in the genome of hybrid plants.     

白桦反义CCoAOMT基因调控木质素生物合成

白桦是我国北方重要的造林树种,但其中的高木质素含量严重制约了它作为造纸资源植物的开发利用。本文利用RACE技术获得了白桦咖啡酰辅酶A-3-O-甲基转移酶（CCoAOMT）基因全长ORF序列,并构建了白桦CCoAOMT基因的反义表达载体,通过农杆菌介导法将其导入到白桦中。PCR检测表明反义CCoAOMT基因已整合到白桦的基因组中。对转化植株的半定量PCR检测显示转基因株系的CCoAOMT基因表达量下降;Wiesner染色发现,与野生型相比,转基因植株木质素含量有所下降。对七年生的转基因白桦和野生型对照进行了化学成分分析,结果表明转基因白桦的苯醇抽提物和Klason木质素显著减少,聚戊糖含量升高。上述结果暗示BpCCoAOMT基因参与白桦木质素的合成,反义表达该基因后木质素含量减少,更易于去除。白桦CCoAOMT基因对木质素的合成起重要作用,这为培育低木质素含量的制浆新品种白桦奠定了基础。     

Impact of differential DNA methylation on transgene expression in cotton (Gossypium hirsutum L.) events generated by targeted sequence insertion

Targeted Genome Optimization (TGO) using site‐specific nucleases to introduce a DNA double‐strand break (DSB) at a specific target locus has broadened the options available to breeders for generation and combination of multiple traits. The use of targeted DNA cleavage in combination with homologous recombination (HR)‐mediated repair, enabled the precise targeted insertion of additional trait genes (2mepsps, hppd, axmi115) at a pre‐existing transgenic locus in cotton. Here we describe the expression and epigenome analyses of cotton Targeted Sequence Insertion (TSI) events over generations. In a subset of events, we observed variability in the level of transgene (hppd, axmi115) expression between independent but genetically identical TSI events. Transgene expression could also be differential within single events and variable over generations. This expression variability and silencing occurred independently of the transgene sequence and could be attributed to DNA methylation that was further linked to different DNA methylation mechanisms. The trigger(s) of transgene DNA methylation remains elusive but we hypothesize that targeted DSB induction and repair could be a potential trigger for DNA methylation.     

A horizontally transferred nuclear gene is associated with microhabitat variation in a natural plant population

Horizontal gene transfer involves the non-sexual interspecific transmission of genetic material. Even if they are initially functional, horizontally transferred genes are expected to deteriorate into non-expressed pseudogenes, unless they become adaptively relevant in the recipient organism. However, little is known about the distributions of natural transgenes within wild species or the adaptive significance of natural transgenes within wild populations. Here, we examine the distribution of a natural plant-to-plant nuclear transgene in relation to environmental variation within a wild population. Festuca ovina is polymorphic for an extra (second) expressed copy of the nuclear gene (PgiC) encoding cytosolic phosphoglucose isomerase, with the extra PgiC locus having been acquired horizontally from the distantly related grass genus Poa. We investigated variation at PgiC in samples of F. ovina from a fine-scale, repeating patchwork of grassland microhabitats, replicated within spatially separated sites. Even after accounting for spatial effects, the distributions of F. ovina individuals carrying the additional PgiC locus, and one of the enzyme products encoded by the locus, are significantly associated with fine-scale habitat variation. Our results suggest that the PgiC transgene contributes, together with the unlinked ‘native’ PgiC locus, to local adaptation to a fine-scale mosaic of edaphic and biotic grassland microhabitats.     

Generating Customized Transgene Landing Sites and Multi-Transgene Arrays in Drosophila Using phiC31 Integrase

Transgenesis in numerous eukaryotes has been facilitated by the use of site-specific integrases to stably insert transgenes at predefined genomic positions (landing sites). However, the utility of integrase-mediated transgenesis in any system is constrained by the limited number and variable expression properties of available landing sites. By exploiting the nonstandard recombination activity exhibited by a phiC31 integrase mutant, we developed a rapid and inexpensive method for isolating landing sites that exhibit desired expression properties. Additionally, we devised a simple technique for constructing arrays of transgenes at a single landing site, thereby extending the utility of previously characterized landing sites. Using the fruit fly Drosophila melanogaster, we demonstrate the feasibility of these approaches by isolating new landing sites optimized to express transgenes in the nervous system and by building fluorescent reporter arrays at several landing sites. Because these strategies require the activity of only a single exogenous protein, we anticipate that they will be portable to species such as nonmodel organisms, in which genetic manipulation is more challenging, expediting the development of genetic resources in these systems.     

Towards effective non-viral gene delivery vector

Despite very good safety records, clinical trials using plasmid DNA failed due to low transfection efficiency and brief transgene expression. Although this failure is both due to poor plasmid design and to inefficient delivery methods, here we will focus on the former. The DNA elements like CpG motifs, selection markers, origins of replication, cryptic eukaryotic signals or nuclease-susceptible regions and inverted repeats showed detrimental effects on plasmids’ performance as biopharmaceuticals. On the other hand, careful selection of promoter, polyadenylation signal, codon optimization and/or insertion of introns or nuclear-targeting sequences for therapeutic protein expression can enhance the clinical efficacy. Minimal vectors, which are devoid of the bacterial backbone and consist exclusively of the eukaryotic expression cassette, demonstrate better performance in terms of expression levels, bioavailability, transfection rates and increased therapeutic effects. Although the results are promising, minimal vectors have not taken over the conventional plasmids in clinical trials due to challenging manufacturing issues.     

影响农杆菌介导的植物转基因的因素

农杆菌介导法是植物转基因研究中应用最多的方法,具有转化成本低、效率高、可转移较长基因片段等特点.多种因素影响此遗传转化过程中的转化率,包括外植体类型、植物基因型、外植体的处理、菌株和质粒载体类型、预培养以及培养基成份等方面.