In vivo electroporation and ubiquitin promoter--a protocol for sustained gene expression in the lung

BACKGROUND: Gene therapy applications require safe and efficient methods for gene transfer. Present methods are restricted by low efficiency and short duration of transgene expression. In vivo electroporation, a physical method of gene transfer, has evolved as an efficient method in recent years. We present a protocol involving electroporation combined with a long-acting promoter system for gene transfer to the lung. METHODS: The study was designed to evaluate electroporation-mediated gene transfer to the lung and to analyze a promoter system that allows prolonged transgene expression. A volume of 250 microl of purified plasmid DNA suspended in water was instilled into the left lung of anesthetized rats, followed by left thoracotomy and electroporation of the exposed left lung. Plasmids pCiKlux and pUblux expressing luciferase under the control of the cytomegalovirus immediate-early promoter/enhancer (CMV-IEPE) or human polyubiquitin c (Ubc) promoter were used. Electroporation conditions were optimized with four pulses (200 V/cm, 20 ms at 1 Hz) using flat plate electrodes. The animals were sacrificed at different time points up to day 40, after gene transfer. Gene expression was detected and quantified by bioluminescent reporter imaging (BLI) and relative light units per milligram of protein (RLU/mg) was measured by luminometer for p.Pyralis luciferase and immunohistochemistry, using an anti-luciferase antibody. RESULTS: Gene expression with the CMV-IEPE promoter was highest 24 h after gene transfer (2932+/-249.4 relative light units (RLU)/mg of total lung protein) and returned to baseline by day 3 (382+/-318 RLU/mg of total lung protein); at day 5 no expression was detected, whereas gene expression under the Ubc promoter was detected up to day 40 (1989+/-710 RLU/mg of total lung protein) with a peak at day 20 (2821+/-2092 RLU/mg of total lung protein). Arterial blood gas (PaO2), histological assessment and cytokine measurements showed no significant toxicity neither at day 1 nor at day 40. CONCLUSIONS: These results provide evidence that in vivo electroporation is a safe and effective tool for non-viral gene delivery to the lungs. If this method is used in combination with a long-acting promoter system, sustained transgene expression can be achieved.     

小鼠胎脑组织活体转基因技术的建立

目的:建立实用的小鼠活体脑组织基因转染技术。方法:将EGFP质粒(CAGS启动子)注射到胎龄16 d(E16d)的胎鼠侧脑室,用镊形电极隔着子宫壁夹住胎鼠头部,在45 V电压下给予5次电脉冲刺激,每次刺激50ms,间隔1 s;转染后不同天数将胎鼠脑组织完整取出,以4%PFA固定后冰冻切片,进行激光共聚焦照相。结果:EGFP质粒被转入小鼠活体脑组织细胞中并获得表达,动物存活率为90%,GFP阳性率高于80%。结论:通过对麻醉剂、电脉冲刺激、质粒浓度、术中术后处理等多种实验条件的摸索,建立了实用的小鼠胎脑组织活体转基因技术。     

A platform of high-efficiency nonviral gene transfer in mouse osteoblast cells in vitro

We have previously established mouse genetic models and identified the genetic components of quantitative trait loci (QTL) on mouse chromosomes that contribute to phenotypes such as bone size, bone density, and bone's anabolic response to mechanical loading. However, these regions contain dozens of unknown genes that are needed for functional testing. In this study, we provided a protocol of nucleoporation with high efficiency by using a commercial nucleofection buffer and Gene Pulser to deliver a test gene into bone cells for functional studies. We cloned an osteoblast differentiation-specific geneosterix (Osx) from a mouse bone cDNA library into a pHGCX expression vector and used nucleoporation to deliver pHGCX/Flag-Osx into the nuclei of MC3T3-E1 cells. We then examined the transfection efficiency transgene expression, and function. Our results have demonstrated that nucleoporation can deliver a transgene into MC3T3-E1 osteoblast cells with approx 94% transfection efficiency, and express a functional Flag-Osx fusion protein capable of inducing cell differentiation as measured by an incease in alkaline phosphatase (ALP) activity. Therefore, this experimental system provides a rapid, safe, and efficient cell-based model of high-throughput phenotypic screening to identify candidate genes from physically mapped regions that are important for osteoblast differentiation.     

Study on the electro-transformation conditions of improving transformation efficiency for Bacillus subtilis

Aims: To optimize the transformation conditions and improve the transformation efficiency of Bacillus subtilis WB800 and DB104. Methods and Results: Trehalose, which could decrease the damage of electric shock to the cells, was added to the electroporation medium containing sorbitol and mannitol. The factors affecting the transformation efficiency, such as the growth phase of bacteria, cell concentration, electric field strength and plasmid variety, were examined and improved. The new method increased the transformation efficiency of B. subtilis by nearly 100‐fold compared with the conventional one. Conclusions: With the optimized method, the transformation efficiency came up to 3·64 × 10⁵ transformants μg^?1 DNA for WB800, and 2·10 × 10⁵ transformants μg^?1 DNA for DB104. Significance and Impact of the Study: This improvement in transformation efficiency will be largely attributed to the research of expression of exogenous genes in B. subtilis, gene library construction for directed evolution and transformation of wild‐type B. subtilis strains.     

Genetic and Genomic Toolbox of the Chordate Ciona intestinalis

The experimental malleability and unique phylogenetic position of the sea squirt Ciona intestinalis as part of the sister group to the vertebrates have helped establish these marine chordates as model organisms for the study of developmental genetics and evolution. Here we summarize the tools, techniques, and resources available to the Ciona geneticist, citing examples of studies that employed such strategies in the elucidation of gene function in Ciona. Genetic screens, germline transgenesis, electroporation of plasmid DNA, and microinjection of morpholinos are all routinely employed, and in the near future we expect these to be complemented by targeted mutagenesis, homologous recombination, and RNAi. The genomic resources available will continue to support the design and interpretation of genetic experiments and allow for increasingly sophisticated approaches on a high-throughput, whole-genome scale.     

电穿孔介导小干扰RNA高效转染小鼠附植前胚胎

使用Cy3标记的阴性对照小干扰RNA(siRNA)转染小鼠附植前胚胎,建立向小鼠附植前胚胎导入siRNA的电穿孔方法。通过控制透明带弱化程度、电压、脉冲时间和脉冲次数等条件,采用不同参数组合并结合使用不同介质作为电转缓冲液将Cy3标记的阴性对照siRNA转染小鼠附植前胚胎。在荧光倒置显微镜下,观察胚胎的存活率、siRNA转染率以及阳性转染存活胚胎的囊胚发育率。结果显示小鼠附植前胚胎在使用台氏液消化胚胎透明带10 s后,以opti-MEM作为电转缓冲液,电穿孔参数设置为30 V,1 ms,3次的条件下取得最佳转染效果。总之,电穿孔方法可实现siRNA简便、高效地转染小鼠附植前胚胎。     

Introduction of silencing-inducing transgene against Fgf19 does not affect expression of Tbx5 and beta3-tubulin in the developing chicken retina

Fgf19 is known to be expressed in the developing chicken eye but its functions during retinal development have remained elusive. Since Fgf19 is expressed in the dorsal portion of the optic cup, it is intriguing to know whether FGF19 is required for expression of dorso-ventral morphogenetic genes in the eye. To clarify this, expression patterns of Tbx5 and Vax were examined in the developing eye after in ovo RNA interference targeted against Fgf19 . Quantitative polymerase chain reaction (PCR) analysis showed that the short-hairpin RNAs (shRNAs) targeted against Fgf19 could reduce its expression in the eye to less than 50% of a relative amount of mRNA, compared with contralateral or untreated control eyes. However, no obvious alteration in expression domains of Tbx5 or Vax was observed. Misexpression of Tbx5 or Tbx5 -RNAi did not alter the Fgf19 expression either. Furthermore, although Fgf19 is expressed in the central retina before neurogenesis occurs, β3-tubulin, a marker for early retinal differentiation was still detected in the central retina after knockdown of Fgf19 . Thus, knockdown of Fgf19 supports no obvious regulations between Fgf19 and Tbx5 , or exhibits no phenotypes that perturb early retinal differentiation.     

Gene application with in utero electroporation in mouse embryonic brain

Mouse genetic manipulations, such as the production of gene knock-out, knock-in, and transgenic mice, have provided excellent systems for analysis of numerous genes functioning during development. Nevertheless, the lack of specific promoters and enhancers that control gene expression in specific regions and at specific times, limits usage of these techniques. However, progress in in utero systems of electroporation into mouse embryos has opened a new window, permitting new approaches to answering important questions. Simple injection of plasmid DNA solution and application of electrical current to mouse embryos results in transient area- and time-dependent transfection. Further modification of the technique, arising from variations in types of electrodes used, has made it possible to control the relative size of the region of transfection, which can vary from a few cells to entire tissues. Thus, this technique is a powerful means not only of characterizing gene function in various settings, but also of tracing the migratory routes of cells, due to its high efficiency and the localization of gene expression it yields. We summarize here some of the potential uses and advantages of this technique for developmental neuroscience research.     

植物体细胞原生质体遗传转化研究

重点介绍了植物体细胞原生质体遗传转化的方法和当前已经取得的成果,同时提出了目前原生质体遗传转化中存在的问题,展望了今后的工作重点。植物原生质体遗传转化的方法主要有：PEG介导转化法、电击穿孔转化法、脂质体介导转化法、农杆菌共培养转化法等。