显微注射构建烟粉虱的RNAi技术体系

RNAi已经成为揭示基因功能的一个常用的有力工具。在昆虫RNAi技术体系中,通过显微注射方法向昆虫体内注入dsRNA,实现系统性干扰,是最为广泛采用的方法。但对于体型较小的昆虫,注射法操作存在较大的难度,特别是非常微小的昆虫烟粉虱Bemisia tabaci。为了进行烟粉虱大规模基因功能验证,需要开发一套成熟的RNAi技术体系。本研究以烟粉虱的CYP6CM1基因为目标基因,通过显微注射法注射不同浓度的dsRNA到烟粉虱4龄若虫,荧光定量PCR检测其沉默效果。结果表明:在对单头烟粉虱注射50 n L时,注射不同浓度的dsRNA,对CYP6CM1基因的沉默效果不同,最大沉默效率可达到80%以上。因此,本实验成功构建了烟粉虱的RNAi技术体系,也可用于其它粉虱类害虫的RNAi技术。     

Analysis of control elements for position‐independent expression of human α‐lactalbumin YAC

A major problem in the production of transgenic animal bioreactors using microinjections is the low production rate of high‐expressing transgenic animals due to the position effect. We previously reported that transgenic rats carrying the 210 kb yeast artificial chromosome (YAC) including the human α‐lactalbumin gene express the transgene in a position‐independent manner. The 210 kb YAC was thought to have all the elements necessary for position‐independent expression. In this paper, we constructed fragmented YAC clones and a cosmid clone, and produced transgenic rats to analyze these elements. Transgenic rats with both the 50 kb upstream and downstream regions of the α‐lactalbumin gene had position‐independent expression. Transgenic rats with the 20 kb upstream and downstream regions, however, had position‐dependent expression. Therefore, all the elements necessary for position‐independent expression are thought to be located in the 50 kb upstream to 50 kb downstream region of the α‐lactalbumin gene. Furthermore, we replaced the human α‐lactalbumin promoter with the bovine αS1‐casein promoter in the 210 kb YAC and produced transgenic rats. Position‐dependent expression was observed. The elements required for position‐independent expression of the bovine αS1‐casein gene are different from those required for the human α‐lactalbumin gene, despite the fact that the two genes have the same tissue and developmental specificity. Mol. Reprod. Dev. 54:17–23, 1999. © 1999 Wiley‐Liss, Inc.     

Overexpression of Fyn tyrosine kinase causes abnormal development of primary sensory neurons in Xenopus laevis embryos

The expression and function of the Src family protein tyrosine kinase Fyn in Xenopus laevis embryos have been examined. In situ hybridization analysis demonstrated nervous system-specific expression of Fyn mRNA in tail-bud embryos. However, a class of primary sensory neurons; that is, Rohon-Beard (RB) neurons, which is positive for immunoglobulin superfamily cell adhesion molecules (CAM), neural cell adhesion molecule (N-CAM) and contactin, is devoid of Fyn expression. Injection of Fyn mRNA into one of the blastomeres at the 2-cell stage led to overexpression of Fyn in the injected half of the tail-bud embryos. Immunolabeling of the embryos with anti-HNK-1 antibody revealed that the peripheral axons of RB neurons were partially misguided and bound to each other to form abnormal subcutaneous fascicles. Similar abnormality was induced by injection of the Fyn overexpression vector. The incidence of abnormality appeared dose-dependent, being 68-92% of the injected embryos at 50-400 pg of mRNA. Co-injection of the contactin antisense vector depleted contactin mRNA accumulation without affecting Fyn overexpression and reduced the incidence of the abnormal RB-cell phenotype. However, the N-CAM antisense was ineffective in reducing this abnormality. These results suggest that Fyn can modify signals regulating axonal guidance or fasciculation in the developing X. laevis nervous system and that contactin may affect this action of Fyn.     

Integration of Controlled Intracellular Pressure Microinjection, lontophoresis, and Membrane Potential Measurement

Abstract: A novel device for intracellular microinjection was designed to integrate controlled pressure microinjection and electrical microinjection (iontophoresis) with membrane poten tial recording and a limited capacity for turgor measurement. The validity of the device was verified by microinjection of a mixture of the fluorescent probes, Texas Red sulfonyl chloride and 10 kDa-LYCH-dextran conjugate, into epidermal cells of var iegated Coleus blumei leaves. Continuous monitoring of the fluorochrome movement by confocal laser scanning microscopy evidenced that the novel device succeeded in differential micro-injection of the fluorescent probes by pressure and iontophor esis. The multifunctionality of the microinjection system was further demonstrated by showing that both microinjection methods functioned in parallel with cellular membrane poten tial measurements in Vicia faba stem tissue. Advantages and prospective applications of the integrated microinjectionjmem-brane potential measurement system are briefly discussed.     

Delivery of dsRNA for RNAi in insects: an overview and future directions

Abstract RNA interference (RNAi) refers to the process of exogenous double‐stranded RNA (dsRNA) silencing the complementary endogenous messenger RNA. RNAi has been widely used in entomological research for functional genomics in a variety of insects and its potential for RNAi‐based pest control has been increasingly emphasized mainly because of its high specificity. This review focuses on the approaches of introducing dsRNA into insect cells or insect bodies to induce effective RNAi. The three most common delivery methods, namely, microinjection, ingestion, and soaking, are illustrated in details and their advantages and limitations are summarized for purpose of feasible RNAi research. In this review, we also briefly introduce the two possible dsRNA uptake machineries, other dsRNA delivery methods and the history of RNAi in entomology. Factors that influence the specificity and efficiency of RNAi such as transfection reagents, selection of dsRNA region, length, and stability of dsRNA in RNAi research are discussed for further studies.     

家蚕转基因方法的初步研究

为建立家蚕转基因研究中切实可行的外源基因导入方法、分别用显微注射法、精子介导法、脂质体法和压力渗透法将含有绿色荧光蛋白(gfp)基因的转座子载体和辅助质粒转入到家蚕的受精卵中。在后代中检测到发绿色荧光的蚕茧,用PCR方法检测到后代个体染色体中含有gfp基因,并比较了上述几种方法的优缺点,为进一步进行转基因家蚕的研究奠定了基础。     

显微注射法制备转基因家蝇技术的建立

旨在利用显微注射法对早期家蝇(Musca domestica L.)卵注射含有增强型绿色荧光蛋白(Enhanced green fluorescent protein,EGFP)基因的转座子载体,实现活体基因稳定表达并对其进行验证,为开展家蝇基因功能的研究奠定基础。文中自制适用于显微注射家蝇卵的硼硅酸盐玻璃微量注射针,摸索出家蝇卵壳的软化处理条件,以NanojectⅢ高精度微量注射器为主体构建适用于家蝇的显微注射技术平台;将含有眼部特异表达的3×P3启动子、EGFP的重组质粒PiggyBac-[3×P3]-EGFP与稳定遗传表达辅助质粒pHA3pig helper显微注射到处理过的家蝇卵中,待羽化观察眼部发光情况,检测EGFP的表达及转录水平。结果表明,将家蝇卵在漂白水中漂洗35 s时卵的正常孵化率为55%,处理35 s的卵壳其硬度适宜注射且注射针头不易破碎;羽化后的家蝇眼部带有绿色荧光的占比约为3%,通过分子检测,家蝇的DNA和RNA中均扩增出EGFP特异片段,大小为750 bp。通过该技术平台,能够便捷、有效地实现报告基因在家蝇中的稳定表达,建立以家蝇为主体的生物反应器,为后续家蝇基因功能的研究提供一定参考价值。     

Production of germ-line chimeras in rainbow trout by blastomere transplantation

We describe a technique for producing germ-line chimeric rainbow trout, Oncorhynchus mykiss, by microinjection of the isolated blastomeres. FITC-labeled donor cells and non-labeled recipient embryos at various developmental stages between the early blastula and early gastrula stages were used for cell transplantation. The chimera formation rate and the degree of donor cell distribution in recipient embryos were evaluated at both the late gastrula stage (5 days post fertilization (dpf)) and the 40-somite stage (10 dpf). Among the six combinations of developmental stages of donor and recipient embryos, the combination of midblastula (2.5 dpf) donor cells and early blastula (1.5 dpf) recipient embryos gave the highest chimera formation rate and the best distribution pattern of donor cells. Using this combination, chimeric rainbow trout were produced with donor blastomeres from dominant orange-colored mutant embryos and wild-type recipient embryos. Of the 238 chimeric embryos produced, 28 (12%) hatched normally and 14 of the 28 fry (50%) had donor-derived orange body color. To test for germ-line transmission of donor cells, gametes obtained from the matured chimeras were fertilized with gametes from wild-type fish. Of the 19 matured chimeras, 6 (32%) yielded donor-derived orange-colored progeny, in addition to wild-type siblings. The contribution rates of donor cells in the germ-line ranged from 0.3 to 14%. This technique for producing germ-line chimeras should be a powerful tool for cell-mediated gene transfer in rainbow trout. Especially, if body color mutants are used for either donor cells or the host embryos, it will be possible to easily concentrate F1 transgenic embryos derived from transplanted donor cells by body color screening. Mol. Reprod. Dev. 59: 380-389, 2001.     

Influence of Heterologous Transplant of DNA‐lacking Mitochondria from Entamoeba histolytica on Proliferation of Entamoeba invadens

In mitochondria, compatibility of proteins encoded in mitochondrial DNA and nuclear DNA is essential for the normal functioning of the organelle. Incompatibility between mitochondrial and nuclear DNA can lead to dysfunctional respiration, mitochondrial diseases, and lethal problems, which suggests that the presence of heterologous mitochondria is unfavorable. In a previous study, we established a transplant method for DNA‐lacking mitochondria (mitosomes) in the anaerobic protozoan Entamoeba histolytica. In this study, interspecies transplant of mitosomes from E. histolytica into Entamoeba invadens, which is a parasitic protozoon of reptiles, was performed using the microinjection method at various temperatures and injection volumes. When E. invadens was used as recipient, it showed higher tolerance to a lower temperature and larger injection volume, in comparison with E. histolytica. After microinjection, donor mitosomes expressing HA‐tag conjugated protein were observed in recipient cells by immunofluorescent staining. The heterologous mitosomes‐injected cells proliferated and growth rate of the microinjected‐cells was similar to that of intact cells. Therefore, we conclude that interspecies transplant of DNA‐lacking mitochondria does not result in incompatibility.     

Transmission of injected DNA sequences to multiple eggs of Metaseiulus occidentalis and Amblyseius finlandicus (Acari: Phytoseiidae) following maternal microinjection

The persistence of DNA injected into two species of adult female phytoseiids and its transmission to serial eggs deposited by them was assessed by the polymerase chain reaction (PCR). The effect of DNA concentration on persistence and transmission was examined in Metaseiulus occidentalis. M. occidentalis females were microinjected with plasmid DNA at three different concentrations (250, 500, 750 ng L^–1) and allowed to deposit one to five eggs before the females and their last eggs were analyzed. Plasmid DNA was found in 82% of the females assayed and in 70% of all the eggs analyzed (including the fifth eggs produced after microinjection). Transmission of DNA to multiple eggs was also examined in Amblyseius finlandicus. Females of this species are less traumatized by microinjection allowing analysis of transmission over a more extended number of eggs. Females were microinjected and allowed to deposit eggs until their death. DNA from every fifth egg was analyzed by the PCR. PCR products were amplified from 51% of the eggs and from all egg classes except the 30th egg. The persistence and presence of plasmid DNA in both eggs and females suggests that (1) maternal microinjection is a more efficient method for DNA delivery than traditional egg microinjection, (2) it may be possible to isolate transformants from fewer maternally-microinjected females than originally expected, and (3) maternal microinjection could be useful as a DNA delivery system in other phytoseiids.