Introduction and Expression of Recombinant Genes in Ascidian Embryos

In order to examine the expression of exogenous genes introduced into ascidian eggs, two recombinant plasmids pmiwZ and pHrMA4aCAT were microinjected into the cytoplasm of fertilized eggs of Ciona savignyi and Halocynthia roretzi , respectively. The plasmid pmiwZ contains the coding sequence of bacterial β-galactosidase gene ( lac-Z ) fused with animal gene promoters, while pHrMA4aCAT was constructed by fusing about 1.4-kb long 5' flanking region of H. roretzi muscle actin gene HrMA4a with bacterial chloramphenicol acetyltransferase gene ( CAT ). Injection of approximately 160 pl of 10 μg/ml pmiwZ DNA into Ciona eggs did not affect the embryogenesis, although introduction of the same volume of 30 μg/ml pmiwZ DNA resulted in abnormal development of injected eggs. When the expression of lac-Z was examined by histochemical detection of the enzyme activity, the expression was evident in the early tailbud embryos and later stage embryos, and larvae, irrespective of linear or circular form of the plasmid. The enzyme activity appeared in various cell-types including epidermis, nervous system, endoderm, mesenchyme, notochord, and muscle. In contrast, when pHrMA4aCAT was introduced into Halocynthia eggs and the appearance of CAT protein was examined later by the anti-CAT antibody, the CAT expression was restricted to muscle cells. These results indicate that the recombinant genes introduced into ascidian eggs could express during embryogenesis and that the 1.4-kb long 5' flanking region of HrMA4a contains regulatory sequences enough for the appropriate spatial and temporal expression of the gene.     

Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato

Larval zebrafish are emerging as a model for describing the development and function of simple neural circuits. Due to their external fertilization, rapid development, and translucency, zebrafish are particularly well suited for optogenetic approaches to investigate neural circuit function. In this approach, light-sensitive ion channels are expressed in specific neurons, enabling the experimenter to activate or inhibit them at will and thus assess their contribution to specific behaviors. Applying these methods in larval zebrafish is conceptually simple but requires the optimization of technical details. Here we demonstrate a procedure for expressing a channelrhodopsin variant in larval zebrafish somatosensory neurons, photo-activating single cells, and recording the resulting behaviors. By introducing a few modifications to previously established methods, this approach could be used to elicit behavioral responses from single neurons activated up to at least 4 days post-fertilization (dpf). Specifically, we created a transgene using a somatosensory neuron enhancer, CREST3, to drive the expression of the tagged channelrhodopsin variant, ChEF-tdTomato. Injecting this transgene into 1-cell stage embryos results in mosaic expression in somatosensory neurons, which can be imaged with confocal microscopy. Illuminating identified cells in these animals with light from a 473 nm DPSS laser, guided through a fiber optic cable, elicits behaviors that can be recorded with a high-speed video camera and analyzed quantitatively. This technique could be adapted to study behaviors elicited by activating any zebrafish neuron. Combining this approach with genetic or pharmacological perturbations will be a powerful way to investigate circuit formation and function.     

A Comparison of GFP‐Tagged Clathrin Light Chains with Fluorochromated Light Chains In Vivo and In Vitro

Clathrin triskelia consist of three heavy chains and three light chains (LCs). Green fluorescent protein (GFP)‐tagged LCs are widely utilized to follow the dynamics of clathrin in living cells, but whether they reflect faithfully the behavior of clathrin triskelia in cells has not been investigated yet thoroughly. As an alternative approach, we labeled purified LCs either with Alexa 488 or Cy3 dye and compared them with GFP‐tagged LC variants. Cy3‐labeled light chains (Cy3‐LCs) were microinjected into HeLa cells either directly or in association with heavy chains. Within 1–2 min the Cy3‐LC heavy chain complexes entered clathrin‐coated structures, whereas uncomplexed Cy3‐LC did not within 2 h. These findings show that no significant exchange of LCs occurs over the time–course of an endocytic cycle. To explore whether GFP‐tagged LCs behave functionally like endogenous LCs, we characterized them biochemically. Unlike wild‐type LCs, recombinant LCs with a GFP attached to either end did not efficiently inhibit clathrin assembly in vitro, whereas Cy3‐ and Alexa 488‐labeled LC behaved similar to wild‐type LCs in vitro and in vivo. Thus, fluorochromated LCs are a valuable tool for investigating the complex behavior of clathrin in living cells.     

Cloning of Black Carp β-actin Gene and Primarily Detecting the Function of Its Promoter Region

A 3 338 bp DNA fragment including the open reading frame and 5′-flanking region of β-actin gene for black carp genome was obtained through PCR amplification. Analysis of the sequencing results indicated the ORF of black carp β-actin gene encoding a 375 amino acid protein that shares a high degree of conservation to other known actins. The black carp β-actin sequence showed 100% identity to common carp, grass carp, and zebrafish, 99.2% identity to human and Norway rat β-actin gene, 98.9% and 98.1% identity to chicken and Kenyan clawed frog β-actin gene, respectively. The promoter region of black carp β-actin gene was inserted into the promoterless pEGFP₁ vector. The recombinant plasmid was microinjected into the fertilized eggs of mud loach before two-cell stage as well as transfected into HeLa cell line. GFP expression was found in 50% of mud loach embryos and 2/3 HeLa cells. The GFP expression could be observed in every part of the mud loach embryos, and in some embryos, the GFP was expressed in the whole body. Thus, the usefulness of black carp β-actin promoter as a ubiquitous expression promoter was confirmed using the EGFP as a reporter gene.     

Microinjection of Guanine NucleotideAnalogues into Lily Pollen Tubes Results in Isodiametric TipExpansion

Abstract: Heterotrimeric and small G-proteins aresupposed to participate in tip growth of plant cells. Quantitative changes intip growth rate after introduction of non-hydrolysable guanine nucleotideanalogues (NA) into lily pollen tubes have been interpreted as support for thehypothesis that heterotrimeric G-proteins regulate tube elongation (Ma et al.,1999 IDREF="R243-11">11). Here, we report that microinjection of guanineNA into lily pollen tubes causes loss of growth polarity, resulting inisodiametric tip swelling. Our results are compatible with current modelssuggesting an involvement of plant Rho-related small G-proteins (Rop) in themaintenance of pollen tube polarity and in tip morphogenesis.     

改良型大鼠鞘内穿刺置管针及导管末端注药装置在大鼠鞘内置管术中的应用研究

目的: 介绍一种大鼠鞘内穿刺置管针及导管末端注药装置在大鼠鞘内置管术中的应用,并评价其可行性和有效性。方法: 60只清洁级SD雄性大鼠,随机分为常规组(C组,n=30)和改良组(M组,n=30)。C组使用尖端磨平的20G针头穿刺置管,置入微量导管后于大鼠颈部两耳间穿出,留置2～3 cm,导管末端热封后游离放置;M组使用鞘内穿刺置管针进行操作,并以微量注射旋塞保护鞘内导管末端。术后1 d评估两组大鼠运动功能,分别于术后1、3、7、14、21 d进行利多卡因试验,至30 d鞘内注射亚甲蓝定位,观察并记录鞘内导管位置;记录两组鞘内给药操作时间;测定术前及术后上述时点两组大鼠机械刺激缩足反应阈值 (PWT);术前及术后一周行旷场实验,评估两组大鼠自主活动情况。结果: 运动功能分级:C组Ⅰ级75.9%,Ⅱ级20.7%,Ⅲ级3.4%,M组Ⅰ级96.7%,Ⅱ级3.3%,M组运动功能分级Ⅰ级占比率显著高于C组(P＜0.05);利多卡因实验和亚甲蓝定位导管位置显示:C组分别于置管后第14日、21日各有1例脱落,至置管后第30日,共有4例导管脱落,而M组导管均在位,两组相比有统计学差异(P＜0.05);M组鞘内注药操作时间多在1 min左右,而C组超过3 min,两组相比有显著统计学差异(P＜0.01);PWT变化情况:两组置管后一周内PWT有波动,第3日降至最低,两组与术前相比均有显著差异(P＜ 0.05),置管后7 d逐渐恢复至术前水平,两组间比较无差异;旷场实验:M组置管前后在总路程、中央区活动路程以及运动时间和速度等方面均无差异,两组间各指标之间相比也无差异。结论: 改良型大鼠鞘内穿刺置管针较常规穿刺置管工具穿刺效能一致,但对大鼠运动功能影响小;微量注射旋塞能有效避免鞘内导管脱出,快速便捷完成注药,并对大鼠自主活动无影响,值得进一步推广使用。     

Association of Antioxidant Systems in the Protection of Human Fibroblasts Against Oxygen Derived Free Radicals

The protection of human diploid fibroblasts against high oxygen tension was investigated using various combinations of the three major antioxidant enzymes: superoxide dismutase, catalase and gluthathione peroxidase. α-Tocopherol, a well-known hydrophobic antioxidant, was also tested in combination with the different enzymes. Microinjection of solutions containing different combinations of the three enzymes was compared with the injection of each single enzyme. We observed that the protections given by catalase or superoxide dismutase on the one hand, and by glutathione peroxidase on the other hand, were additive. Surprisingly, the combinations of catalase and superoxide dismutase were less effective than catalase alone and was even toxic at low SOD concentrations. Addition of α-tocopherol following the injection of any of the three enzymes was highly beneficial, but the strongest synergistic effect was obtained with glutathione peroxidase. These results stress the importance of membrane protection by α-tocopherol and indirectly by glutathione peroxidase. They also showed that any injection leading to the decrease in the O₂^?. or H₂ O ₂ concentration combined with one of these two protectors is very beneficial for the cells probably by decreasing the OH concentration. This is also proven by the very good protective effect obtained with desferrioxamine.     

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.     

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing ¹. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth ^2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes ^4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development⁶.The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos⁷. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells^8-10. We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol¹¹. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.     

线虫基因显微注射和整合技术——设备、操作与指南

秀丽线虫(Caenorhabditis elegans)是研究动物遗传、个体发育和行为活动的重要模式生物,其主要优点是能够研究从分子细胞水平到整体系统水平的相关生命活动的作用机理．其中基因显微注射和整合是该领域的核心技术,广泛应用于研究线虫的基因表达、功能和基因间的相互作用等．显微注射技术使用尖端开口直径为微米级的玻璃微管,将所研究的目的DNA注射进线虫的性腺．注射的DNA被成熟的卵细胞吸收,以染色体外遗传物质的形式存在．但这种形式并不能稳定遗传,可采用基因整合技术将外源基因整合到染色体上,得到稳定遗传的线虫种系．显微注射是一项精细的实验技术,影响实验成功与否的因素较多,实际操作需要有一定的经验．在实践过程中逐步改进和完善了这项技术,在此主要介绍线虫显微注射技术的操作过程、创新方法以及注意细节．