Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant. The technique consists of intravenous microinjections on 2 dpf zebrafish. This technique represents an efficient and rapid method to deliver soluble substances into the bloodstream of zebrafish larvae, allowing for the injection of 15-20 fish per hour. In addition to AKI studies, this microinjection technique can also be used for other types of experimental studies such as angiography. We provide a detailed protocol of the technique from equipment required to visual measures of decreased kidney function. In addition, we also demonstrate the process of fixation, whole mount immunohistochemistry with a kidney tubule marker, plastic embedding and sectioning of the larval zebrafish. We demonstrate that zebrafish larvae injected with gentamicin show morphological features consistent with AKI: edema, loss of cell polarity in proximal tubular epithelial cells, and morphological disruption of the tubule.     

家蚕卵显微注射操作对蚕卵孵化及畸形蚕发生的影响

早期胚胎显微注射是目前获得转基因家蚕Bombyx mori的主要途径。显微注射操作对蚕卵的损伤导致注射后的蚕卵孵化率降低, 是家蚕转基因工作的主要障碍之一。本研究对不同卵龄的蚕卵进行了开孔或注射实验, 并对产后5 h的蚕卵上背侧、 腹侧、 前极、 后极和中央等5个不同的位置进行了开孔实验, 调查了卵孵化率和体形异常蚕的产生情况。结果表明： 较早卵龄期的注射或从蚕卵背侧的注射可以获得高的孵化率。腹侧注射产生大量的体形异常蚕而背侧注射的蚕完全正常。通过调整注射时期和注射位置避开上述影响可以减少死卵和畸形蚕, 提高孵化率。本研究为改进家蚕转基因操作技术提供了有效的参考。     

显微注射青霉素G获得单感染Wolbachia和单感染Cardinium白背飞虱品系

Wolbachia和Cardinium都是广泛存在于节肢动物体内的一类母系遗传的共生细菌, 可以通过不同方式操纵寄主的生殖行为。Wolbachia和Cardinium感染同一寄主在自然界比较常见, 但是在某些可以同时感染Wolbachia和Cardinium的寄主中其单感染品系较难发现。本研究检测了云南文山(YN)、 海南三亚(HN)这2个不同地理种群中Wolbachia和Cardinium的感染情况; 以双感染Wolbachia和Cardinium的白背飞虱Sogatella furcifera海南种群为实验材料, 运用显微注射方法对双感染Wolbachia和Cardinium的白背飞虱若虫注射不同浓度青霉素G以获得单感染品系。结果表明： 白背飞虱自然种群中单感染Wolbachia比率极低, 本实验用到的海南种群未检测到单感染Wolbachia成虫; 通过显微注射青霉素G的方法可以从白背飞虱双感染品系中筛选获得单感染品系, 当青霉素G注射浓度为0.2%(w/v), 注射龄期为5龄时得到单感染品系效率最高; F5代的检测结果显示显微注射得到的单感染品系可以稳定遗传。本研究为获得单感染品系白背飞虱提供了快捷方法, 同时为其他双感染Wolbachia和Cardinium节肢动物不同感染品系的筛选提供参考。     

Generation of Transgenic Hydra by Embryo Microinjection

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology¹. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.     

The Involvement of PLC-IP3 Signaling Pathway in Pollen Tube Growth

The effect of phospholipase C (PLC) signaling pathway on lily (Lilium davidii Duch.) pollen tube elongation was examined by means of microinjection. Pollen tube elongation was inhibited by microinjecting antibodies against animal PLCβ1-3 or inositol-1,4,5-triphosphate receptor （IP3R2, 3）, but was not affected by antibodies against animal PLCβ4 or IP3R1. Pollen tube elongation was also stimulated significantly by microinjecting IP3. The results suggest that PLC-IP3 signaling pathway might present in pollen system and be involved in pollen tube growth.     

氯化钠密度梯度离心法制备用于显微注射的外源DNA片段

DNA显微注射是生产转基因动物最可靠和最常使用的一种方法,外源DNA的纯度对显微注射的成功起着至关重要的作用。本文介绍用氯化钠密度梯度离心的方法制备用于显微注射的外源DNA片段。与传统的琼脂糖凝胶回收的方法相比较,用此方法制备的外源DNA片段对小鼠受精卵进行显微注射后,受卵体母鼠的胚胎存活率,以及子代小鼠的外源基因整合率均有明显的提高。这一方法可为进一步提高转基因动物的成功率,提供方法学上的参考。 Abstract:DNA microinjection is the most popular and reliable method of producing transgenic animals.The purity of foreign DNA plays an important role for the success of microinjection.In this study,we introduced the use of sodium chloride step gradients in fractionating foreign DNA fragment for microinjection.The data demonstrated that,compared with the conventional agarose gel extraction method,NaCl purification scheme of toreign DNA could improve the treated embryo survival and foreign DNA intergration rate markedly.     

Metabolic Turnover of Fatty Acids and Acylglycerols in Rat Sciatic Nerve

To explain the discrepancy between the low level and high metabolic activity of endoneurial free fatty acids (FFAs) and triacylglycerol (TG), levels of de novo synthesized FFA and acylglycerols were measured in rat sciatic endoneurium at various intervals after endoneurial microinjection of [14C]acetate. Soon after injection (less than 10 min), the [14C]acetate was metabolized to FFA and incorporated into diacylglycerol (DG), TG, sterols, ceramides, and various phospholipids. The proportions of 14C-labeled FFA, DG, TG, and ceramides to total 14C-labeled lipids decreased, whereas those of phospholipids and cerebrosides increased with time after injection. These findings suggest that rapid turnover of FFA and TG may contribute to their low level in sciatic endoneurium. The de novo synthesized fatty acids were largely incorporated into phosphatidylcholine (approximately 50% of total 14C-labeled phospholipids), probably via the cytidine nucleotide pathway using 1,2-DG as a metabolic intermediate. Hydrolysis of [14C]phosphatidylcholine revealed that fatty acids were labeled at both the C-1 (approximately 43%) and C-2 (approximately 57%) positions. On the other hand, a temporal association between decreased amounts of 14C-label in ceramides and increased amounts of 14C-label in sphingomyelin and galactocerebrosides supports the hypothesis that peripheral nerve galactocerebroside is derived, in vivo, from ceramide via acylation of sphingosine. This exclusive labeling of endoneurial lipids by endoneurial microinjection of labeled precursor provides a unique model for studying synthesis and metabolic turnover of membrane lipids in experimental neuropathies.     

紫竹梅雄蕊毛发育过程中胞间连丝通透能力的动态变化

运用显微注射法将不同分子量的荧光分子探针注入紫竹梅雄蕊毛细胞中,在荧光显微镜下观察其通过胞间连丝转移情况。表明,开放花和花蕾Ⅳ期胞间连丝允许通过物质的分子量不超过１０００Ｄ,衰老花可允许ＦＩＴＣ－ｉｎｓｕｌｉｎＡ链（２９２１Ｄ）通过,而花蕾Ⅰ期可允许ＦＩＴＣ－ｄｅｘｔｒａｎ（４４００Ｄ）通过。说明胞间连丝允许通过物质的分子量极限不是固定不变的,它随组织、细胞的发育进程而改变。     

大鼠延髓腹面尾端加压区与头端加压区心血管效应的差异

雄性Ｗｉｓｔａｒ大鼠５６只,乌拉坦麻醉,双侧脑区微量注射谷氨酸钠,观察延髓腹面尾端加压区（ｃＶＭＰ）与头端加压区（ｒＶＭＰ）心血管效应的差异。结果表明：ｃＶＭＰ的升压效应弱于ｒＶＭＰ,不伴随明显的心率效应,ｒＶＭＰ升压效应强于ｃＶＭＰ兼有明显的心率增加。在谷氨酸钠兴奋作用下,ｒＶＭＰ抑制动脉压力感受反射,而ｃＶＭＰ易化该反射。提示两者对心血管功能的影响具有不同的神经途径和机能意义。