基于鸡胚电转技术研究脊髓神经干细胞相关基因功能方法的建立

目的建立一种基于鸡胚电转技术研究脊髓神经干细胞(NSCs)相关基因功能的方法。方法 RT-PCR检测鸡胚发育不同时期脊髓NSCs表面标志物;在鸡胚胚龄(E)E2.5~E3时,利用活体电转基因技术将p CAGGSGFP质粒转染到鸡胚脊髓,E6时体视荧光显微镜下筛选绿色荧光蛋白(GFP)阳性胚胎,每组至少取材5个;通过脊髓横切及open-book技术观察神经纤维投射情况;普通光学显微镜下剥离出3~5条脊髓,经胰蛋白酶消化、离心后,无血清NSCs培养基重悬获得细胞铺板,于37℃、5%CO_2细胞培养箱内培养,定时观察GFP阳性脊髓NSCs的形态变化。结果 RT-PCR结果表明,鸡胚脊髓中阳性表达NSCs表面标志物;随后的脊髓横切及open-book结果表明,GFP阳性转染侧的神经纤维能穿过底板,投射到脊髓对侧;而脊髓NSCs体外培养结果显示,GFP标记的脊髓细胞具有典型的NSCs形态,继续培养后有明显突起产生。结论本实验成功建立了一种基于鸡胚电转技术研究脊髓神经干细胞相关基因功能的方法。     

A simple technique for early in vivo electroporation of E1 chick embryos

BACKGROUND: The amenability of the chick embryo to a variety of manipulations has made it an ideal experimental model organism for over 100 years. The ability to manipulate gene function via in ovo electroporations has further revolutionized its value as an experimental model in the last 15 years. Although in ovo electroporations are simple to conduct in embryos ≥ E2, in ovo electroporations at early E1 stages have proven to be technically challenging due to the tissue damage and embryonic lethality such electroporations produce. RESULTS AND CONCLUSIONS: Here we report our success with in vivo microelectroporations of E1 embryos as young as Hamburger-Hamilton Stage 4 (HH4). We provide evidence that such electroporations can be varied in size and can be spatially targeted. They cause minimal disruption of tissue-size, 3-dimensional morphology, cell survival, proliferation, and cell-fate specification. Our paradigm is easily adapted to a variety of experimental conditions since it does not depend upon the presence of a lumen to enclose the DNA solution during electroporation. It is thus compatible with the in vivo examination of E1 morphogenetic events (e.g., neural tube closure) where preservation of 3-dimensional morphology is critical.     

In ovo exposure to monochromatic lights affect posthatch muscle growth and satellite cell proliferation of chicks: role of IGF-1

To study the role of IGF-1 on stimulation with monochromatic light during incubation altering posthatch muscle growth, chicken embryos were exposed to blue light, green light, red light, white light or darkness throughout embryonic period and then were raised in white light conditions upon hatching. Comparing with the other treatment groups, the chicks in green light group had heavier hatching weights, higher muscle indexes and larger muscle fibers. Both in vivo and in vitro studies showed that the number and proliferative activity of satellite cells in green light group were the highest. Plasma IGF-1 level and skeletal muscle IGF-1R mRNA level were higher in green light group. Moreover, exogenous IGF-1 increased the proliferative activity of satellite cell in a dose-dependent fashion. These results suggest that stimulation with monochromatic green light during incubation promoted posthatch muscle growth and satellite cell proliferation of chicks through IGF-1 signaling.     

Electroporation as a technique for the transfer of macromolecules into mammalian cell lines

Summary Numerous methods have been devised to facilitate the introduction of exogenous compounds into cells. The technique of electroporation allows the direct physical transfer of numerous kinds of molecules into essentially any cell, but the major application has been for transfection of DNA and this emphasis is recapitulated here. However, the conditions for transfer of DNA or other macromolecules are sufficiently similar that the same protocol is followed regardless. In addition, as electroporation involves a mechanism distinct from that of most other methods of transfection, it has distinct advantages and disadvantages relative to other transfection techniques. This review is designed to allow one to simplify the processes of determining whether electroporation is appropriate to a given experimental design, to indicate how to minimize the disadvantages, and to simplify the requisite process of parameter optimization required to evaluate and apply electroporation to the system of choice. Practical aspects are highlighted but the theoretical bases are discussed when relevant for application of the technique.     

鼠胚大脑皮层子宫内电穿孔法基因转移体系的建立

体内电穿孔法转基因技术作为一种新型有效的转基因方法,被广泛应用于发育生物学的研究,特别是中枢神经系统发育。本研究旨在通过子宫内电穿孔法,建立鼠胚大脑皮层体内基因转移体系:将带有GFP的表达载体pCAGGS-IRES-EGFP显微注入胚胎期14.5d的鼠胚侧脑室中,电极沿与脑中缝平行的方向夹持鼠脑,在电压30V,脉冲时间50ms,脉冲数5的条件下方波电击,将外源基因转移到室管膜层细胞。胚胎被重新放回母体内,继续发育3d。分离被转染的鼠胚脑组织,沿冠状面冰冻切片,在荧光显微镜下观察到外源蛋白GFP在鼠胚大脑皮层中的瞬时表达。鼠胚大脑皮层子宫内电穿孔法基因转移体系的建立将为进一步研究皮层发育、神经元迁移、轴突导向等以及神经发生过程中相关基因功能奠定基础。     

Parameters affecting efficiency of in ovo electroporation of the avian neural tube and crest

Background: Many variations in avian in ovo transfection of the neural tube/crest have been reported, but never compared quantitatively. Results: Genome integrating pT2K‐CAGGS‐GFP and pCAGGS‐T2TP transposase plasmids were co‐electroporated into quail E2 embryo trunk neural tube and the proportion of GFP‐expressing neural cells was counted 1 and 7 days later. Electroporation efficiency increased with plasmid concentration and pulse number but plateaued at, respectively, above 1.25 µg/µL and 3 pulses. Bilateral electroporation transfected more cells than unilateral but less than that anticipated by doubling the unilateral treatment. Holding the concentration of GFP plasmid constant and varying the transposase plasmid concentration revealed an optimum ratio of, in this case, 4:1 (1.2 µg/µL:0.3 µg/µL). Leaving transfected embryos to E9 confirmed that expression was maintained in vivo with the transposase system, but declined with non‐integrated plasmid. Transfection of neural crest cells was low if electroporated less than 6–8 hr before emigration. We propose this indicates loss of epithelial integrity well prior to exit. We suggest this event be termed epithelio‐mesenchymal transition sensu stricto, whereas the term delamination be reserved for the later emigration from the neural epithelium. Conclusions: Co‐electroporation in ovo must take into account plasmid(s) concentration and ratio, pulse number, pulse directionality, and timing. Developmental Dynamics 243:1440–1447, 2014. © 2014 Wiley Periodicals, Inc.     

电穿孔技术的研究及应用进展

脉冲电场的电穿孔效应在医学上的应用是一门多学科的生物医学工程学技术，已证实其主要的生物学效应是使靶细胞膜发生可逆及不可逆性电击穿。近年电穿孔效应所介导的电化学治疗应用广泛。对电脉冲的量—效关系、电场分布、膜穿标记物及生物电阻成像等基础研究及临床应用中的进展及尚存问题作一综述。     

电脉冲介导基因在小鼠杂交瘤细胞的高效转移

本文应用电脉冲介导基因转移法,获得pSV2-neo基因在小鼠杂交瘤细胞(B889)中的高效转移和稳定表达。研究了电场强度、脉冲宽度、脉冲次数等电场参数对基因转化频率的影响和小鼠杂交瘤细胞最适电转移条件。     

电穿孔技术的研究及应用进展

脉冲电场的电穿孔效应在医学上的应用是一门多学科的生物医学工程学技术,已证实其主要的生物学效应是使靶细胞膜发生可逆及不可逆性电击穿。近年电穿孔效应所介导的电化学治疗应用广泛。对电脉冲的量—效关系、电场分布、膜穿标记物及生物电阻成像等基础研究及临床应用中的进展及尚存问题作一综述。     

电穿孔疗法在头颈癌治疗中的应用

头颈癌严重地威胁着人类生命 ,而传统的化学治疗方法由于受癌细胞对药物吸收率的限制 ,其疗效也受到限制。电穿孔由于能使细胞膜出现暂时微孔 ,从而能大大提高癌细胞对药物的吸收率。在此基础上 ,结合传统的化疗方法形成的电穿孔疗法成为一种新型的头颈癌治疗法。博来梅素由于其自身的优良特性成为电穿孔疗法的首先药物。离体实验和临床研究表明 ,与传统方法相比 ,电穿孔疗法对于治疗头颈癌能取得更好的疗效     

In ovo non‐invasive quantification of the myocardial function and mass of chick embryos using magnetic resonance imaging

Magnetic resonance imaging (MRI) has evolved as one of the major non‐invasive tools to study healthy and diseased hearts in animal models, especially rodent models. Even though, the chick embryo has long been used as a model for cardiovascular research, MRI has not yet been used for in vivo cardiac studies. Part of the reason for this is the difficulty in monitoring the ECG and respiration of the chick embryo in the magnet for gating purposes. To overcome this complication, this paper presents the use of retrospective Cine MRI to measure the cardiac function of chick embryos in ovo for the first time, without the need for respiratory or cardiac gating. The resulting left ventricular functional parameters, from six chick embryos at 20 days of incubation, were (mean ± SD) EDV 69 ± 15 µL, ESV 31 ± 7 µL, SV 38 ± 9 µL and EF 54.5 ± 2%. The use of retrospective Cine MRI at earlier stages of development is also discussed and difficulties have been highlighted. Copyright © 2009 John Wiley & Sons, Ltd.     

电穿孔增强核酸疫苗免疫反应的研究进展

核酸疫苗自诞生以来在肿瘤、感染性疾病和自身免疫性疾病等方面显示出其独特的优越性。目前,研究如何增强核酸疫苗的免疫原性是一大热点,在众多方法中,电穿孔通过增加细胞对DNA的摄取,使表达的目的抗原增多,从而明显地增强了核酸疫苗诱导的免疫反应。就电穿孔技术在核酸疫苗研究领域的应用作一综述。     

小鼠子宫内胚胎电转技术方法的改良

目的 优化小鼠子宫内胚胎电转技术,用于检测胎鼠大脑新生神经元的迁移和分化特征。 方法 通过选择异氟烷气体麻醉方式,改进毛细玻璃针的制备方法,减少手术中胚胎的暴露时间,优化电转参数等,对小鼠子宫内胚胎电转技术进行一系列优化。采用优化的子宫内胚胎电转技术给6只怀孕16.5 d的孕鼠进行了手术,共电转胚胎42只。 结果 电转后孕鼠和胚胎的存活率达到100%,其中大脑表达外源基因的胎鼠占85%±3%。 结论 优化的小鼠子宫内胚胎电转技术可以获得令人满意的胎鼠存活率和基因电转效率。     

Generation of monoclonal antibodies against Blo t 3 using DNA immunization with in vivo electroporation

BACKGROUND: House dust mite allergy is closely associated with allergic diseases. Blomia tropicalis mite species is an important clinical species in the tropics. The cDNA clone encoding Blo t 3, a group 3 allergen from B. tropicalis, has been isolated in our laboratory. OBJECTIVE: This study was designed to generate Blo t 3-specific monoclonal antibodies (mAbs) for the detection, characterization and purification of this allergen. METHODS: Mice were immunized intramuscularly with naked plasmid DNA encoding Blo t 3 gene with in vivo electroporation. Hybridomas were generated by the fusion of the splenocytes to X63-Ag8.653 myeloma cells. Purified native Blo t 3 was obtained by mAb immuno-affinity purification and the allergenicity of native Blo t 3 was determined by human IgE enzyme-linked immunosorbent assay (ELISA). RESULTS: A panel of class-switched and high-affinity mAb recognizing a wide spectrum of Blo t 3 epitopes have been generated. These mAbs are useful for western immunoblot assay, sandwich ELISA and affinity purification of native Blo t 3. Allergenicity of native Blo t 3 protein was examined with 44 mite-allergic sera and approximately 57% of the tested sera had positive serum IgE reactivity to the native Blo t 3. CONCLUSIONS: These results demonstrated that intramuscular injection of naked DNA encoding Blo t 3 gene combined with in vivo electroporation is an effective and simple method to raise monoclonal antibodies that can be used for characterization and purification of Blo t 3.     

不同时期转染基因对牵引区新骨生成的影响

背景：前期研究表明,电穿孔介导的重组质粒pIRES-hBMP2-hVEGF165能促进牵引区早期新生血管的形成和新骨形成。 目的：观察不同时机转染基因对兔下颌骨牵张成骨过程中牵引区新骨生成的影响,探索基因导入的最佳转染时间,以获得更好的治疗效果。 方法：新西兰大白兔48只,全麻下行双侧下颌骨截骨及牵引器植入后,采用随机区组法分成4组,分别于造模后即刻、牵引开始时、牵引结束时在双侧牵引区注射2 μg(0.1 g/L)重组质粒pIRES-hBMP2-hVEGF165,3组均予电穿孔刺激;单纯牵引组单纯牵引不行基因转染。各组于造模后3 d开始以0.8 mm/d、1次/d的速率进行牵引,连续牵引10 d;各组分别于固定期1,2,4,8周处死3只兔子,切取下颌牵引区新生组织行组织学检测和形态计量学分析。 结果与结论：组织学检查和形态计量学分析发现,牵引期转染组与即刻转染组、固定期转染组、单纯牵引组比较间隙内有更多的新生血管、成骨细胞和间充质细胞等成分,各时点新生骨量与新生骨小梁宽度明显高于后者(P < 0.05)。表明在牵引开始时(牵引期)进行基因转染较其他时间转染促进新骨生成作用明显,能够获得最佳的促进新骨生成的效果,提示牵引期是下颌骨基因治疗的最佳时机。     

Mitotic patterns in the migrating lateral line cells of zebrafish embryos

The sense organs of the posterior lateral line system (neuromasts) are formed by a migrating primordium. In zebrafish, the primordium comprises approximately 100 cells at the onset of migration, and has deposited approximately 300 cells by the end of the process. Here, we report localized phases of mitotic activity and of mitotic quiescence within the migrating primordium. Quiescence in the leading region seems associated to the formation of a new prospective neuromast, whereas quiescence in the trailing region follows a wave of mitoses that synchronize trailing cells in G0/G1 phase, anticipating neuromast differentiation. Manipulating the size of the primordium does not lead to changes in the rate of cell proliferation. We also show that two mitoses often take place nearly synchronously in adjacent cells, suggestive of a determinate lineage. We conclude that proliferation in the migrating primordium follows a stereotyped pattern that closely anticipates the normal development of the system. Developmental Dynamics 238:1042–1051, 2009. © 2009 Wiley‐Liss, Inc.     

一种自制电极鸡胚视顶盖转基因技术

目的建立一种高效鸡胚视顶盖电转基因技术,对自制电极进行评估。方法在鸡胚发育的第5天(E5),将0.5 g/L的p CAGGS-绿色荧光蛋白(GFP)质粒0.25~0.5μl准确地注射到视顶盖内,每组60个鸡胚,在电压18V、每次脉冲60ms、间隔100ms、电脉冲6次的条件下,以原装电极为对照,自制电极为实验组进行定时定位活体电转基因,电转后在E6~E12时观察胚胎成活率,取材后在体视荧光显微镜下观察报告基因GFP表达结果以此判断转染的效率。结果在鸡胚模型中,通过自制电极转染鸡胚视顶盖,24h后鸡胚成活率达到89%,在鸡胚发育到E12时存活率达到35%,与原装电极相比分别提高48.3%和600%个百分点。结论自制电极在鸡胚视顶盖电转过程对胚胎的损伤小,转染后胚胎成活率高,为鸡胚视顶盖电转基因提供一种高效的转染技术。     

The effects of agouti-related protein gene transfer in vivo by electroporation in mice

In the present study, the cDNA encoding agouti-related protein (AGRP) gene known as an orexigenic factor was transferred in vivo to test whether food intake and body weight gain is improved in mice. When the expression plasmid of AGRP gene driven by mouse β-actin, pActAGRP, was transferred into leg muscle by electroporation, body weight of gene-transferred mice was significantly increased at 14 days and afterwards compared with that of control counterparts (p < 0.05). Likewise, daily food intake was also significantly higher in the AGRP gene-transferred mice than in the control mice at 4 days and afterwards (p < 0.05). A significant increase in serum AGRP concentration of the AGRP gene-transferred group was detected compared with the control group at 1 week (p < 0.01), but the difference quickly disappeared at 3 weeks. However, the hypothalamic NPY mRNA abundance of AGRP gene-transferred mice was significantly higher than that of the control mice at 3 weeks (p < 0.05). These results suggested that instead of hormone administration per se, in vivo AGPR gene transfer into skeletal muscle was found to mimic hormonal effects. The present methodology of in vivo gene transfer by electroporation might be useful to promote growth and food intake in farm livestock as well as experimental animals.     

Detection and localization of avian alphaherpesviruses in embryonic tissues following in ovo exposure

We investigated whether chicken embryonic tissues are susceptible to infection with virulent Marek’s disease virus (MDV). Groups of embryonic day (ED) 17 chicken embryos and 1-day-old chicks were compared for tissue sites of viral persistence of MDV and herpesvirus of turkeys (HVT) in lungs, thymuses, bursae of Fabricius and spleens. MDV DNA was detectable in the lungs and thymuses of embryos at 3 days post-inoculation (DPI) by in situ hybridization, while HVT DNA was only present in embryonic lungs. The target cells in lungs and thymuses appeared non-lymphoid and lymphoid, respectively. By 5 days post-inoculation, both viruses were detectable in all organs examined and persisted after hatch. Although MDV DNA was present in the embryo, there was little evidence of viral replication. These findings demonstrate the differences in pathogenesis of embryonic infection with MDV and HVT and provide evidence that the chicken embryo is susceptible to infection with a virulent avian herpesvirus.