Efficient gene silencing induction in tomato by a viral satellite DNA vector

Virus-induced gene silencing (VIGS) in tomato (Lycopersicon esculentum Mill.) is currently routinely analysed using Tobacco rattle virus (TRV)-based vector. We recently reported a new vector system modified from DNA beta (DNAm beta) of Tomato yellow leaf curl China virus (TYLCCNV) for VIGS analysis in Solanaceous species including tomato. Here, we describe DNAm beta-induced gene silencing in tomato. We found that DNAm beta-induced gene silencing was initiated from vascular tissues, and later scattered to other tissues. Once initiated in seedlings, the silencing phenotype lasted for the entire life span of the plants, was expressed in a variety of tissues and organs including leaf, shoot, stem, flower and fruit, and could be achieved at any growth stage. It was insensitive to temperature as high as 32 degrees C and no symptoms were observed in silenced plants. The DNAm beta vector worked efficiently in at least seven tomato cultivars, indicating that this system has great potential as a versatile VIGS system for routine functional analysis of genes in tomato.     

Bovine viral diarrhea virus is a suitable viral vector for stable expression of heterologous gene when inserted in between N(pro) and C genes

Bovine viral diarrhea virus (BVDV) is a group of small enveloped viruses with a single-stranded, positive-oriented RNA genome of approximately 12.3 kb. BVDV genome directs the production of a viral polyprotein that is subsequently cleaved to release the mature viral proteins. To explore the potential of using BVDV as viral vector for stable expression of heterologous genes, eGFP2A was inserted in between N(pro) and C genes of a noncytopathic type-I BVDV strain SD1. eGFP2A was designed with eGFP protein in frame fused to the N terminus of the foot-and-mouth disease virus 2A protease. This strategy promised not only the correct processing of both viral N(pro) and C protein but also releasing of the chimeric protein from the nascent viral polyprotein. The recombinant reporter virus was successfully rescued in MDBK cells. In vitro study showed that eGFP2A protein, as expected, was expressed and processed properly from the nascent viral polyprotein. The reporter virus was similar to wt SD1 in viral RNA replication and protein expression and comparable to wt SD1 in growth kinetics except that this virus had a peak virus titer approximately 0.5 log(10) lower and a maximum yield about 4h later than wt SD1. In summary, these results indicated that BVDV is a suitable viral vector for stable expression of heterologous genes when inserted in between N(pro) and C genes.     

Adenovirus expression vector mediated gene silencing in suspension cells

目的 利用重组腺病毒载体携带短发夹RNA (shRNA),使难于转染的悬浮细胞达到较高的目的基因沉默效率.方法 分别将含有人胎盘生长因子(PLGF)的干扰序列及阴性对照序列(scramble)连入pRNAT-H1.1/Adeno载体,获得重组穿梭质粒;将线性化的重组穿梭质粒与腺病毒骨架重组;将重组子转染HEK 293细胞,经过3轮病毒包装,收获病毒颗粒并测定滴度.病毒颗粒感染悬浮细胞——人小细胞肺癌细胞(NCI-H 250和NCI-H 209),通过实时定量PCR方法鉴定靶基因沉默效应,并利用肿瘤细胞重建基质膜侵袭实验进行功能鉴定.结果 成功构建带有靶基因干扰序列及scramble、空载体的3种腺病毒重组子(AD-shuttle-shPLGF、AD-shuttlescramble和AD-shuttle).经过HEK 293细胞包装,最终获得3株病毒颗粒,病毒滴度分别为1.5×1011、1.6×1011和1.6×1011.NCI-H 250和NCI-H 209细胞感染腺病毒颗粒48 h后,大部分细胞表达GFP,感染率接近100％;且两株细胞感染AD-shuttle-shPLGF病毒48 h后,PLGF mRNA表达水平降低(P＜0.01),肿瘤细胞侵袭能力明显下降(P＜0.01).结论 携带shRNA的腺病毒表达载体可以代替电转、脂质体介导等方法,使难于转染细胞的目的基因达到有效沉默.     

利用腺病毒载体介导的RNA干扰技术在悬浮细胞中获得基因沉默效应

 摘要： 目的 利用重组腺病毒载体携带短发夹RNA （short hairpin RNA,shRNA）,使难于转染的悬浮细胞达到较高的目的基因沉默效率。方法 将含有人胎盘生长因子（placental growth factor,PLGF）干扰序列的76 nt的寡核苷酸序列连入含有绿色荧光蛋白基因（GFP）的腺病毒穿梭质粒pRNAT-H1.1/Adeno中,获得重组穿梭质粒。重组穿梭质粒经限制性内切酶PmeⅠ线性化、去磷酸化回收后与腺病毒骨架pAdeasy-1共电转入BJ5183感受态细胞。同源重组后经限制性内切酶PacⅠ酶切鉴定,挑选阳性克隆并大量获得重组质粒。PacⅠ酶切质粒后转染HEK 293细胞,经过三轮病毒包装,收获病毒颗粒,经OD260方法和TCID50方法测定病毒滴度。病毒颗粒感染悬浮细胞——人小细胞肺癌细胞（NCI-H 250和NCI-H 209）,通过实时定量PCR方法鉴定靶基因沉默效应,并利用肿瘤细胞重建基质膜侵袭实验进行功能鉴定。结果 NCI-H 250和NCI-H 209细胞在感染腺病毒颗粒48 h后,大部分细胞表达GFP,感染率接近100%;两株细胞感染腺病毒48 h后,PLGF mRNA表达水平明显下降,且靶基因沉默的肿瘤细胞侵袭能力明显下降。结论 携带shRNA的腺病毒表达载体,可以代替电转、脂质体介导等方法,使难于转染细胞的目的基因达到有效沉默。     

A modified Sindbis vector for prolonged gene expression in neurons

Sindbis viruses have been widely used in neurobiology to express a variety of genes in cultured neurons, in cultured slices, and in vivo. They provide fast onset and high levels of expression of foreign genes, but the expression is limited to a short time window due to a shut-off of host protein synthesis. We have used a mutation in an essential gene (nsP2) of the life cycle of Sindbis, which allows the functional analysis of changes in protein expression for >/=6 days after infection. This Sindbis mutant (nsP2) was used to express enhanced green fluorescent protein (EGFP) in hippocampal neurons in culture and in vivo without any sign of toxicity, based on two-photon imaging and electrophysiology. In addition, the EGFP mutant virus can be injected in vivo to visualize spines and other details of neuronal structure. The Sindbis mutant described here provides an improved tool in neurobiology with reduced cytotoxicity and a prolonged time window of expression for novel applications in imaging and behavior. In addition, the use of this vector for the functional expression of mammalian voltage-gated ion channels in organotypic slices is demonstrated.     

DNA abrasion onto plants is an effective method for geminivirus infection and virus-induced gene silencing

Geminiviruses belong to a rapidly growing group of plant pathogens that contribute to crop losses in tropical and subtropical areas of the world. Geminivirus infection is a model for plant DNA replication and virus/host interactions. Geminiviruses are also used as vectors to induce silencing of endogenous genes in several plant species. A method was analyzed for inoculating geminiviruses using plasmid DNA rubbed onto leaves in the presence of an abrasive (DNA abrasion). Although the use of DNA abrasion to inoculate geminiviruses has been described previously, the technique has fallen out of favor and has not been systematically optimized. However, consistent efficiencies of 100% infection rates can be achieved by DNA abrasion. The symptoms of Tomato Golden Mosaic Virus or Cabbage Leaf Curl Virus infection on Nicotiana benthamiana were similar in timing and appearance to the symptoms observed in plants inoculated using Agrobacterium as the delivery method. More importantly, silencing of an endogenous gene was highly efficient when a geminivirus silencing vector was inoculated by the DNA abrasion method. Other plant species successfully inoculated with geminiviruses by DNA abrasion were Nicotiana tabacum, Capsicum annuum and Nicandra physalodes. Unfortunately, Arabidopsis thaliana could not be infected with Cabbage Leaf Curl Virus using leaf abrasion, demonstrating limitation of the method. However, leaf abrasion to inoculate geminiviruses is an easy and inexpensive method that should be considered as an accessible technique to the growing number of researchers using geminiviruses.     

A transformation vector for stage-specific expression of heterologous genes in Trypanosoma cruzi epimastigotes

To express heterologous genes in a stage-specific manner, we constructed a transformation vector for Trypanosoma cruzi containing a selection gene (hyg) and a reporter gene (luc) flanked by sequences of the multicopy 1f8 gene arranged so as to provide a trans-splicing acceptor site to hyg and a putative polyadenylation signal to luc. The intergenic region of the T. cruzi genes 294 and KAP was placed between hyg and luc, contributing the polyadenylation signal of 294 to hyg and the KAP trans-splicing acceptor site to luc. Transformation was carried out by electroporation, and transformed epimastigotes were selected in medium containing hygromycin B. Through double homologous recombination of the 1f8 sequences with their chromosomal counterparts, the construction is inserted into the 1f8 locus, substituting probably one and no more than a few copies of the 1f8 gene without having apparent deleterious effects on the parasite. cDNA analysis demonstrated that the introduced signals were correctly processed, resulting in translatable hyg and luc mRNAs. Whereas epimastigotes express luciferase, no expression is found in the trypomastigote stage.     

Barley stripe mosaic virus (BSMV) as a virus-induced gene silencing vector in maize seedlings

Barley stripe mosaic virus (BSMV) was the first reported and still widely used virus-induced gene silencing (VIGS) vector for monocotyledons including wheat and barley. Despite BSMV’s reported infectivity on maize (Zea mays), the use of the virus as a vector in maize has not been optimized. Here, we assayed infectivity of BSMV in different maize cultivars by vascular puncture inoculation. Through knockdown of the endogenous host phytoene desaturase gene, we demonstrate for the first time that BSMV can be used as a VIGS vector in maize. This adds BSMV to the repertoire of tools available for functional studies in maize.     

shRNA表达载体pWH1的构建及用于HIF1基因的沉默

目的:构建用于RNAi的shRNA(small hairpin RNA)表达载体及检测其对低氧诱导因子-1(Hypoxia-inducible Factor-1,HIF1)基因的沉默效果.方法:从人血基因组中PCR扩增出H1基因启动子,克隆入酶切处理后的pEGFP-C1载体片段中,此载体命名为pWH1.以人HIF1 cDNA基因为靶标设计引物,退火后克隆入pWH1.新的载体转染SGC7901细胞,然后用RT-PCR和Western blot检测HIF1基因的表达改变.结果:构建的pWH1载体能很好地表达针对HIF1基因的shRNA,RT-PCR和Western blot的结果显示HIF1基因的mRNA和蛋白表达水平均明显下降.结论:成功构建了shRNA表达载体pWH1,这对于基因的功能研究具有重要的意义.     

The multifunctional plant viral suppressor of gene silencing P19 interacts with itself and an RNA binding host protein

Tomato bushy stunt virus (TBSV) is an RNA plant virus encoding a protein of approximately 19 kDa (P19) that is involved in various activities important for pathogenicity, including virus transport and suppression of gene silencing. In this study, we provide evidence in vivo and in vitro that P19 specifically interacts with itself to predominantly form dimers, and with a novel host protein, Hin19. Hin19 has a high degree of similarity with a class of RNA-binding proteins of which many are involved in RNA processing. The binding of P19 to itself and to Hin19 both depend on a structurally important central region of P19 that was previously shown critical for its biological function in plants. Our findings provide evidence for a model in which virus spread through suppression of defense-related gene silencing involves the formation of a complex that includes P19 dimers and a newly identified host RNA-binding protein.     

DNA-based mutation analysis of Bruton's tyrosine kinase gene in patients with X-linked agammaglobulinaemia

The identification of the BTK (Bruton's tyrosine kinase) genedefective in human immunoglobulln deficiency X-linked agammaglobulinaemla(XLA) and characterlsation of BTK exon–intron boundarleshas now allowed the analysis of mutations and polymorphismsat the level of genomic DNA. Using Southern blot analysis andthe polymerase chain reaction single strand conformation polymorphism(PCR–SSCP) assay, amplifying all 19 exons and the putativepromoter region with a single annealling temperature, mutationshave been identified in 19 out of 24 unrelated patients diagnosedas having XLA. Apart from a large deletion involving exon 19,nine missense (F25S, R288W, I370M, M509V, R525P, N526K, R562W,A582V and G594R), two nonsense (E277X and R525X), five frameshiftand two splice site mutations have been found affecting mostcoding exons and all major enzyme domains. No mutations or polymorphismswere detected in the putative promoter region. A single nucleotidedeletion located in the last exon, resulting in a truncationof the eight C-terminal residues of Btk and a typical XLA phenotype,indicates structural and/or functional importance of Btk helixI In the catalytic domain. Although allelic heterogeneity atthe BTK locus may partly explain clinical variability In familleswith XLA, compensatory and redundant mechanisms involved inB-cell development must play a role in the phenotypic diversityof the disease.     

A modified GEWF solution is cost-saving and effective for lymph node retrieval in resected colorectal carcinoma specimens

Lymph node (LN) retrieval is important for proper staging of colorectal carcinoma. Although various assistant techniques were recommended to facilitate LN identification, most of them were unavoidably time-consuming, resource intensive and costly.     

Sandwiching of a gene within 12 kb of a functional telomere and alpha satellite does not result in silencing

Pericentrlc heterochromatin and telomeres have been shown tobe capable of repressing the expression of genes located Inclose proximity. The effect of adjacent structural sequenceson gene expression will be important in the design of mammalianartificial chromosomes. In the process of using telomere-containlngconstructs to generate a deletion panel of the long arm of thehuman X chromosome, several cell lines were produced which appearedby in situ hybridization to be broken In Xq at or near the centromere.After analysis of end clones rescued from these cell lines,only two produced data consistent with breaks In the alpha satellitearray without accompanying rearrangements. The mitotic stabilityof an X chromosome, with at least 750 kb of the alpha satellitearray deleted, was compared to controls where the alpha satellitearray remained Intact. No significant change In the stabilityof the chromosome was observed, suggesting that the truncatedchromosome has a fully functional mitotic centromere. Therewas no detectable change in the expression of the hygromycinresistance gene, which is located between a functional centromereand telomere, In this cell line. This study indicates that structuralelements flanking a mammalian selectable marker do not resultin silencing.     

Bhendi yellow vein mosaic disease in India is caused by association of a DNA Beta satellite with a begomovirus

Yellow vein mosaic disease is the major limitation in the production of bhendi or okra (Abelmoschus esculentus), an important vegetable crop of India. This disease is caused by a complex consisting of the monopartite begomovirus Bhendi yellow vein mosaic virus (BYVMV, family: Geminiviridae) and a small satellite DNA beta component. BYVMV can systemically infect bhendi upon agroinoculation but produces only mild leaf curling in this host. DNA beta induces typical symptoms of bhendi yellow vein mosaic disease (BYVMD) when co-agroinoculated with the begomovirus to bhendi. The DNA beta component associated with BYVMD has a number of features in common with those reported for ageratum yellow vein disease and cotton leaf curl disease. BYVMV represents a new member of the emerging group of monopartite begomoviruses requiring a satellite component for symptom induction.     

The UL4 protein of equine herpesvirus 1 is not essential for replication or pathogenesis and inhibits gene expression controlled by viral and heterologous promoters

Defective interfering particles (DIP) of equine herpesvirus 1 (EHV-1) inhibit standard virus replication and mediate persistent infection. The DIP genome is comprised of only three genes: UL3, UL4, and a hybrid gene composed of portions of the IR4 (EICP22) and UL5 (EICP27) genes. The hybrid gene is important for DIP interference, but the function(s) of the UL3 and UL4 genes are unknown. Here, we show that UL4 is an early gene activated solely by the immediate early protein. The UL4 protein (UL4P) was detected at 4 hours post-infection, was localized throughout the nucleus and cytoplasm, and was not present in purified virions. EHV-1 lacking UL4P expression was infectious and displayed cell tropism and pathogenic properties in the mouse model similar to those of parental and revertant viruses. Reporter assays demonstrated that the UL4P has a broad inhibitory function, suggesting a potential role in establishing and/or maintaining DIP-mediated persistent infection.     

DNA immunization in combination with effective antiretroviral drug therapy controls viral rebound and prevents simian AIDS after treatment is discontinued

DNA immunization in conjunction with antiretroviral therapy was evaluated in SIV-infected rhesus macaques treated with [R]-9-[2-phosphonylmethoxypropyl]adenine (PMPA). Macaques were immunized monthly with DNA vaccines expressing either SIV gag/tat or SIV gag/tat and 19 CD8+ T cell epitopes during 7 months of therapy. Half the animals from each group were additionally immunized before infection. Only 60% of the animals (4 controls, 20 vaccinated) responded to PMPA (ART responders). All 4 ART responder controls demonstrated viral rebound or CD4 decline after PMPA was withdrawn. In contrast, 17 of 20 vaccinated ART responders contained viral rebound for over 7 months after PMPA was withdrawn. Viral control correlated with stable CD4 counts, higher lymphoproliferation and an increase in the magnitude and breadth of the CD8+ T cell response. Immunizing before infection or with multi-epitopes enhanced these effects. These results demonstrate that DNA immunization during antiretroviral therapy may be an effective strategy to treat HIV infection.