Characterization of three loci for homologous gene targeting and transgene expression

Integrative gene transfer is widely used for bioproduction, drug screening, and therapeutic applications but usual viral methods lead to random and multicopy insertions, contribute to unstable transgene expression and can disturb endogenous gene expression. Homologous targeting of an expression cassette using rare‐cutting endonucleases is a potential solution; however the number of studied loci remains limited. Furthermore, the behavior and performance of various types of gene cassettes following gene targeting is poorly defined. Here we have evaluated three loci for gene targeting, including one locus compatible with the proposed Safe Harbor criteria for human translational applications. Using optimized conditions for homologous gene targeting, reporter genes under the control of different promoters were efficiently inserted at each locus in both sense and antisense orientations. Sustainable expression was achieved at all three loci without detectable disturbance of flanking gene expression. However, the promoter, the integration locus and the cassette orientation have a strong impact on transgene expression. Finally, single targeted integrations exhibited greatly improved transgene expression stability versus multicopy or random integration. Taken together, our data suggest a potential set of loci for site‐specific transgene integration, suitable for a variety of biotechnological applications. Biotechnol. Bioeng. 2013; 110: 2225–2235. © 2013 Wiley Periodicals, Inc.     

Site‐specific T–DNA integration in Arabidopsis thaliana mediated by the combined action of CRE recombinase and ϕC31 integrase

Random T–DNA integration into the plant host genome can be problematic for a variety of reasons, including potentially variable transgene expression as a result of different integration positions and multiple T–DNA copies, the risk of mutating the host genome and the difficulty of stacking well‐defined traits. Therefore, recombination systems have been proposed to integrate the T–DNA at a pre‐selected site in the host genome. Here, we demonstrate the capacity of the ?C31 integrase (INT) for efficient targeted T–DNA integration. Moreover, we show that the iterative site‐specific integration system (ISSI), which combines the activities of the CRE recombinase and INT, enables the targeting of genes to a pre‐selected site with the concomitant removal of the resident selectable marker. To begin, plants expressing both the CRE and INT recombinase and containing the target attP site were constructed. These plants were supertransformed with a T–DNA vector harboring the loxP site, the attB sites, a selectable marker and an expression cassette encoding a reporter protein. Three out of the 35 transformants obtained (9%) showed transgenerational site‐specific integration (SSI) of this T–DNA and removal of the resident selectable marker, as demonstrated by PCR, Southern blot and segregation analysis. In conclusion, our results show the applicability of the ISSI system for precise and targeted Agrobacterium‐mediated integration, allowing the serial integration of transgenic DNA sequences in plants.     

The sense of agency is action–effect causality perception based on cross-modal grouping

Sense of agency, the experience of controlling external events through one''s actions, stems from contiguity between action- and effect-related signals. Here we show that human observers link their action- and effect-related signals using a computational principle common to cross-modal sensory grouping. We first report that the detection of a delay between tactile and visual stimuli is enhanced when both stimuli are synchronized with separate auditory stimuli (experiment 1). This occurs because the synchronized auditory stimuli hinder the potential grouping between tactile and visual stimuli. We subsequently demonstrate an analogous effect on observers'' key press as an action and a sensory event. This change is associated with a modulation in sense of agency; namely, sense of agency, as evaluated by apparent compressions of action–effect intervals (intentional binding) or subjective causality ratings, is impaired when both participant''s action and its putative visual effect events are synchronized with auditory tones (experiments 2 and 3). Moreover, a similar role of action–effect grouping in determining sense of agency is demonstrated when the additional signal is presented in the modality identical to an effect event (experiment 4). These results are consistent with the view that sense of agency is the result of general processes of causal perception and that cross-modal grouping plays a central role in these processes.     

Spatial summation in the tactile sensory system: Probability summation and neural integration

Psychophysical thresholds for the detection of a 300-Hz burst of vibration applied to the thenar eminence were measured for stimuli applied to the skin through 1.5?cm² and through 0.05?cm² contactors. Thresholds were approximately 13?dB lower when the area of the contactor was 1.5?cm² than when it was 0.05?cm². The difference between the thresholds measured with the large and small contactors was significantly reduced when only the lowest thresholds obtained in the testing sessions were considered. This result supports the hypothesis that one component of spatial summation in the P channel is probability summation. In addition, threshold measurements within a session were less variable when measured with the 1.5?cm² contactor. We conclude that spatial summation in the P channel is a joint function of two processes that occur as the areal extent of the stimulus increases: probability summation in which the probability of exceeding the psychophysical detection threshold increases as the number of receptors of varying sensitivities increases, and neural integration in which neural activity originating from separate receptors is combined within the central nervous system rendering the channel more sensitive to the stimulus.     

城-镇-乡人口教育差距的演变及影响机制

在城乡一体化的过程中,以平均受教育水平测量的城乡人口教育差距,并没有出现库兹涅茨倒U型发展,乡村、县镇与城市之间的差距都在不断扩大。对近20年间差距演变的过程进行分析表明,乡村和县镇人口平均受教育水平的增长速度始终低于城市,尤其是县镇区域几乎没有增长。通过对主要影响因素及作用机制的理论分析和实证检验,研究发现,经济发展水平的提高并没有促使已经存在的差距自然消弥,城乡两个被分割的群体通过显性渠道与隐性渠道不断地文化教育再生产是差距存在和扩大的社会基础;高中阶段教育投入的增加更多是促进了城镇人口受教育水平的提高,初中阶段教育投入城乡差距的扩大直接导致人口受教育程度差距的扩大,而小学阶段教育投入城乡差距没有显示出统计上显著的影响;非户籍人口流动造成城乡人口结构的异质化,并进而扩大城乡常住人口受教育程度的差距;户籍人口迁入对城市和县镇人口受教育程度都有提升作用,迁出在抵消迁入的提升作用后,仍然导致城市与县镇之间教育差距的扩大。     

Integration: An Effective Strategy to Develop Multifunctional Energy Storage Devices

Energy storage devices are arousing increasing interest due to their key role in next‐generation electronics. Integration is widely explored as a general and effective strategy aiming at high performances. Recent progress in integrating a variety of functions into electrochemical energy storage devices is carefully described. Through integration at the level of materials: flexible, stretchable, responsive, and self‐healing devices are discussed to highlight the state‐of‐the‐art multi‐functional electronics. Through the integration at the level of devices, the incorporation of photovoltaic and piezoelectric devices is detailed to reflect the advances in self‐powering electronics. Integrated energy storage devices are presented for wearable applications to indicate a new growth direction. The main challenges and important directions are summarized to offer some useful clues for future development.     

Identifying and retargeting transcriptional hot spots in the human genome

Mammalian cell line development requires streamlined methodologies that will reduce both the cost and time to identify candidate cell lines. Improvements in site‐specific genomic editing techniques can result in flexible, predictable, and robust cell line engineering. However, an outstanding question in the field is the specific site of integration. Here, we seek to identify productive loci within the human genome that will result in stable, high expression of heterologous DNA. Using an unbiased, random integration approach and a green fluorescent reporter construct, we identify ten single‐integrant, recombinant human cell lines that exhibit stable, high‐level expression. From these cell lines, eight unique corresponding integration loci were identified. These loci are concentrated in non‐protein coding regions or intronic regions of protein coding genes. Expression mapping of the surrounding genes reveals minimal disruption of endogenous gene expression. Finally, we demonstrate that targeted de novo integration at one of the identified loci, the 12^th exon‐intron region of the GRIK1 gene on chromosome 21, results in superior expression and stability compared to the standard, illegitimate integration approach at levels approaching 4‐fold. The information identified here along with recent advances in site‐specific genomic editing techniques can lead to expedited cell line development.