Use of a short A/T-rich cassette for enhanced expression of cloned genes inEscherichia coli

A short (43-bp) A/T-rich stretch of DNA located in The intergenic region between thebaiA2 andbaiF genes fromEubacterium sp. strain VPI 12708 was amplified by polymerase chain reaction (PCR) and inserted in front of the Shine-Dalgarno (SD) sequences of three inefficiently-expressedEubacterium sp. strain VPI 12708 genes cloned inEschcrichia coli plasmids. Insertion of this A/T-rich cassette increased gene expression in all cases tested. Deletion of part of the A/T-rich region from abaiF clone in pUC19 resulted in decreased gene expression. Synthesis of specific mRNA was increased with addition of the A/T-rich cassette to constructs containing thebaiC gene from Eubacterium sp. strain VPI 12708, but mRNA synthesis was not significantly changed in cells containing plasmid constructs with thebaiF andbaiG genes. Enhanced translation resulting from a decrease in mRNA secondary structure in the ribosome binding site region is discussed as a possible reason for increased gene expression with the A/T-rich cassette.     

Microinjection of mRNA encoding rat synapsin Ia alters synaptic physiology in identified motoneurons of the crayfish,Procambarus clarkii

Studies of identified neurons have made important contributions to our understanding of cellular neurophysiology. We have developed a technique for modifying gene expression in identified motoneurons of the crayfish Procambarus clarkii in the isolated nervous system as well as in the intact animal through the injection of exogenously synthesized RNAs. mRNA suitable for injection was transcribed in vitro from cDNA templates cloned into a plasmid, pSEM. Initially, mRNAs encoding green fluorescent protein (GFP) and β-galactosidase were injected into the soma of the motor giant neuron (MoG) to determine whether these mRNAs could be successfully translated into protein. Both proteins were expressed. Measurements of GFP fluorescence increase indicated that GFP mRNA was stable and translated into protein for at least 3 days postinjection. We then examined the effects of expression of GFP, AASP-168 (an endogenous crayfish axonal protein), and rat synapsin Ia on MoG synaptic physiology. The mRNA injection procedure did not appear to directly influence synaptic physiology based on the results of the AASP-168 and GFP injections. Injection of mRNA encoding rat synapsin Ia resulted in a significant increase in peak excitatory postsynaptic potential (EPSP) amplitude during repetitive stimulation. These data are consistent with previous studies that have shown that synapsin deficiency reduces synaptic vesicle numbers. The translation of mRNAs with diverse functions and species of origin suggests that this approach will prove useful for studying the function of a wide variety of endogenous and exogenous genes in identified neurons. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 224–236, 1998     

A new strategy for assessing selenoprotein function: siRNA knockdown/knock-in targeting the 3'-UTR

Selenocysteine insertion into protein in mammalian cells requires RNA elements in the 3'-untranslated regions (3'-UTRs) of selenoprotein genes. The occurrence of these conserved sequences should make selenoproteins particularly amenable for knockdown/knock-in strategies to examine selenoprotein functions. Herein, we utilized the 3'-UTR of various selenoproteins to knock down their expression using siRNAs and then knock in expression using constructs containing mutations within the target region. Thioredoxin reductase 1 (TR1) knockdown in a mouse kidney cell line resulted in the cells growing about 10% more slowly, being more sensitive to UV radiation, and having increased apoptosis in response to UV than control cells. The knockdown cells transfected with a construct encoding the wild-type TR1 gene and having mutations in the sequences targeted by siRNA restored TR1 expression and catalytic activity, rendered the knockdown cells less sensitive to UV, and protected the cells against apoptosis. We also applied this technique to other selenoproteins, selenophosphate synthetase 2 and glutathione peroxidase 1, and found that mRNA and protein levels were restored following transfection of knockdown cells with the corresponding knock-in constructs. In addition to important new insights into the functions of key mammalian selenoproteins, the data suggest that the RNAi-based knock-in technology could distinguish phenotypes due to off-targeting and provide a new method for examining many of the subtleties of selenoprotein function not available using RNAi technology alone.     

Brugia malayi: transient transfection by microinjection and particle bombardment

To develop a method for the introduction of DNA into filarial parasites, several methods that have proven successful in other organisms were evaluated for their ability to transform Brugia malayi. Luciferase activity was detectable in embryos bombarded with gold particles coated with a construct consisting of a luciferase reporter gene under the control of the 5S rRNA intergenic spacer (SL promoter). Similar results were seen in adult parasites and infective larvae bombarded with this construct, or in adult female parasites microinjected with the plasmid. In similar experiments employing the SL promoter driving a green fluorescent protein (GFP) reporter, expression of the reporter was detectable in the intrauterine embryos of the microinjected adult parasites, and in the sub-cuticular tissues of biolistically transfected adult female parasites. A similar pattern of GFP expression to that seen in the SL promoter construct transfected parasites was noted in parasites transfected with constructs consisting of the upstream domain derived from an aspartyl aminoacyl tRNA synthetase gene of B. malayi. The ability to transfect B. malayi embryos may provide a foundation for studies of the regulation of gene expression and function in these organisms.     

Expression of endogenous and microinjected hsp 30 genes in early Xenopus laevis embryos

In the present study, we have examined the regulation of expression of a newly isolated member of the hsp 30 gene family, hsp 30C. Using RT-PCR, we found that this gene was first heat-inducible at the tailbud stage of development. We also examined the expression of two microinjected modified hsp 30C gene constructs in Xenopus embryos. One of the constructs had 404 bp of hsp 30C 5′-flanking region, whereas the other had 3.6 kb. Both gene constructs had 1 kb of 3′-flanking region. RT-PCR assays were employed to detect the expression of these microinjected genes. The presence of extensive 5′- and 3′-flanking regions of the hsp 30C gene did not confer proper developmental regulation, since heat-inducible expression of both of the microinjected constructs was detectable at the midblastula stage. The premature expression of the microinjected hsp 30 gene was not a result of high plasmid copy number or the presence of plasmid DNA sequences. These results suggest that the microinjected genes contain all the cis-acting DNA sequences required for correct heat-inducible regulation but do not contain the elements required for the proper regulation of hsp 30 gene expression during development. It is possible that regulatory elements controlling the developmental expression of the hsp30 genes may reside upstream or downstream of the entire cluster. © 1993Wiley-Liss, Inc.     

电穿孔介导质粒DNA肿瘤内转移抑制恶性肿瘤生长与转移

利用携带绿色荧光蛋白（green fluorescent protein, GFP）编码基因的表达质粒,测试电穿孔方法介导目的基因活体组织内转移的效率并优化电击参数.在此基础上采用电穿孔技术直接将编码白介素12（IL-12）、白介素2（IL-2）、粒单细胞克隆刺激因子（GM-CSF）等免疫调节因子或反义血管内皮细胞生长因子121（VEGF₁₂₁）、可溶性血管内皮细胞膜受体（sFlk-1及ExTek）等血管生成抑制因子表达质粒转移至肿瘤局部.实验结果表明电穿孔介导GFP表达质粒肌肉内转移的效率较高,GFP可在肌细胞内持续高水平表达3周以上,而在肿瘤细胞内只能表达4～6 d,但高电压短脉冲电击组肿瘤内GFP阳性细胞数比低电压长脉冲组高2.68倍.多次电击介导IL-12表达质粒转移至肿瘤组织内,可有效地抑制小鼠膀胱癌BTT-gfp、人乳腺癌MCF-7及肝癌SMMC 7721-gfp的生长.MCF-7对血管生成抑制因子基因转移治疗较敏感,单独应用反义VEGF₁₂₁、sFlk-1或ExTek即显示明确的治疗效果.SMMC 7721-gfp单独应用sFlk-1有效.小鼠膀胱癌对单独应用反义VEGF₁₂₁、sFlk-1或ExTek治疗效果不理想,但联合应用sFlk-1和ExTek仍然可以有效地抑制肿瘤生长与转移,甚至使肿瘤缩小或消失.提示电穿孔技术是一项高效、安全、经济的体内基因转移方法.     

Cnidarian and bilaterian promoters can direct GFP expression in transfected hydra

Complete sexual development is not easily amenable to experimentation in hydra. Therefore, the analysis of gene function and gene regulation requires the introduction of exogenous DNA in a large number of cells of the hydra polyps and the significant expression of reporter constructs in these cells. We present here the procedure whereby we coupled DNA injection into the gastric cavity to electroporation of the whole animal in order to efficiently transfect hydra polyps. We could detect GFP fluorescence in both endodermal and ectodermal cell layers of live animals and in epithelial as well as interstitial cell types of dissociated hydra. In addition, we could confirm GFP protein expression by showing colocalisation between GFP fluorescence and anti-GFP immunofluorescence. Finally, when a FLAG epitope was inserted in-frame with the GFP coding sequence, GFP fluorescence also colocalised with anti-FLAG immunofluorescence. This GFP expression in hydra cells was directed by various promoters, either homologous, like the hydra homeobox cnox-2 gene promoter, or heterologous, like the two nematode ribosomal protein S5 and L28 gene promoters, and the chicken beta-actin gene promoter. This strategy provides new tools for dissecting developmental molecular mechanisms in hydra; more specifically, the genetic regulations that take place in endodermal cells at the time budding or regeneration is initiated.     

miR-122过表达转基因小鼠质粒构建及其功能验证

目的:比较两种miR-122转基因小鼠过表达载体构建方法,为建立miR-122过表达转基因小鼠奠定基础。方法:PCR扩增长约291bp的pre-miR-122的序列,分别定向克隆到pBROAD3-GFP载体GFP基因上游内含子或下游3'UTR区域,两种质粒分别转染293T细胞,Q-PCR检测miR-122和GFP的表达水平,并观察GFP绿色荧光。miR-122 sensor reporter是将3个miR-122成熟序列的反义序列串联克隆至psiCHECK2载体luciferase 3'UTR中,然后分别与2种miR-122过表达质粒载体共转染293T细胞,最后检测荧光素酶活性来鉴定miR-122调控功能。结果:2种构建方法的miR-122表达水平都明显增高,而只有插入到GFP基因3'UTR的质粒表达GFP功能正常。结论:构建microRNA过表达载体时,microRNA位于报告基因3'UTR区域不会影响microRNA和报告基因的功能;构建的两种miR-122过表达质粒载体都可应用到转基因小鼠研究中,而将miR-122插入到GFP下游的方法则更利于miR-122的表达。     

Mini-transposon Tn5gfp constructs for differential tagging of microorganisms

Recently thegfp (green fluorescent protein) gene from the jellyfishAequoria victoria has been widely used as a reporter gene. In this study mini-transposons, named as mini-Tn5gfp, were constructed by subcloning thegfp gene into a transposon Tn5. To improve the expression level of thegfp gene, tandom array ofgfp gene was obtained. The constructs were successfully used in tagging target microorganisms by transposition. The level of GFP expression was found to be closely correlated with the copy number of the gfp transposed. These constructs will facillitate not only efficient tagging of whole organism but also genetic marking of target genes by transposition.     

Gene Transfer and Cloning of Flanking Chromosomal Regions Using the Medaka Fish Tol2 Transposable Element

For the ultimate purpose of developing genetic tools using the medaka fish Tol2 transposable element, we examined whether it can transfer a marker gene into the fish genome and also be applied for cloning of chromosomal regions adjacent to insertion points. An internal region of Tol2 was removed and replaced with the green fluorescent protein (GFP) gene and a bacterial plasmid replication origin. This modified Tol2 clone was microinjected into fertilized eggs together with messenger RNA for the Tol2 transposase. The GFP gene was found to be integrated into chromosomes and transmitted to subsequent generations. Restriction enzyme digestion of genomic DNA of a transformant fish, followed by ligation and introduction into bacteria, produced a plasmid containing the entire element and flanking chromosomal regions. Sequencing analysis of this clone demonstrated transposition of the element in the germline of the first generation. Thus, the basic requirements for a gene transfer vector and gene tagging system were fulfilled. Received July 30, 2001; accepted October 4, 2001     

Specific gene silencing using small interfering RNAs in fish embryos

Recently, small interfering RNAs (siRNAs) have been used for gene knockdown in mammalian cultured cells, but their utility in fish has remained unexplored. Here we demonstrate a siRNA-mediated gene silencing technique in rainbow trout embryos. We found that siRNAs effectively suppressed the transient expression of episomally located foreign GFP genes at an early developmental stage and inhibited the expression of GFP genes in stable transgenic trout embryos. Similar gene silencing was observed with an siRNA against the endogenous tyrosinase A gene. siRNAs interfered with the expression of maternally inherited mRNA. siRNAs did not affect non-relevant gene expression and siRNAs with a 4 base mismatch did not affect target gene expression. siRNA gene silencing is therefore highly sequence-specific. Our findings are the first evidence that siRNA-mediated gene silencing is effective in fish. This technique could be a powerful tool for studying gene function during embryonic development in aquacultural fish species, zebrafish, and medaka.     

DNA Oligonucleotides and Plasmids Perform Equally as Donors for Targeted Gene Conversion

Site-specific gene modifications in cells are initiated by the introduction of exogenous DNA. We used a recently established cell assay to compare the ability of DNA donors to induce a single point mutation that converts a target gene encoding blue fluorescent protein (BFP) into expressing green fluorescent protein (GFP). In a chromosomal assay with cells stably expressing BFP, we showed that fluorescently labeled single-stranded oligonucleotides and a donor plasmid cotranscribing a red fluorescent protein provide similar efficiencies in triggering BFP–GFP conversions. In transient cotransfections, an isogenic donor plasmid comprising a nonfunctional GFP gene yielded a greater efficiency for the conversion of the BFP target gene than a nonisogenic donor, and all plasmid donors were superior to oligonucleotides.