Schistosoma mansoni U6 gene promoter-driven short hairpin RNA induces RNA interference in human fibrosarcoma cells and schistosomules

RNA interference (RNAi) mediated by short hairpin-RNA (shRNA) expressing plasmids can induce specific and long-term knockdown of specific mRNAs in eukaryotic cells. To develop a vector-based RNAi model for Schistosoma mansoni, the schistosome U6 gene promoter was employed to drive expression of shRNA targeting reporter firefly luciferase. An upstream region of a U6 gene predicted to contain the promoter was amplified from genomic DNA of S. mansoni. A shRNA construct driven by the predicted U6 promoter targeting luciferase was assembled and cloned into plasmid pXL-Bac II, the construct termed pXL-BacII_SmU6-shLuc. Luciferase expression in transgenic fibrosarcoma HT-1080 cells was significantly reduced 96 h following transduction with plasmid pXL-BacII_SmU6-shLuc, which encodes luciferase mRNA-specific shRNA. In a similar fashion, schistosomules of S. mansoni were transformed with the SmU6-shLuc or control constructs. Firefly luciferase mRNA was introduced into transformed schistosomules after which luciferase activity was analyzed. Significantly less activity was present in schistosomules transfected with pXL-BacII_SmU6-shLuc compared with controls. The findings revealed that the putative S. mansoni U6 gene promoter of 270 bp in length was active in human cells and schistosomes. Given that the U6 gene promoter drove expression of shRNA from an episome, the findings also indicate the potential of this putative RNA polymerase III dependent promoter as a component regulatory element in vector-based RNAi for functional genomics of schistosomes.     

A novel Δ-pyrroline-5-carboxylate synthetase gene of Medicago truncatula plays a predominant role in stress-induced proline accumulation during symbiotic nitrogen fixation

Proline accumulates in environmentally stressed plant cells including those of legume roots and nodules, but how its level is regulated is poorly understood. Δ¹-Pyrroline-5-carboxylate synthetase (P5CS), the committed-step enzyme of proline biosynthesis, is encoded by two duplicated genes in many plants. Here, we isolated MtP5CS3, a third gene, from Medicago truncatula, whose predicted polypeptide sequence is highly similar to those of previously isolated MtP5CS1 and MtP5CS2 except an extra amino-terminal segment. MtP5CS3 was strongly expressed under salinity and drought in shoots and nodulating roots, while MtP5CS1 was constitutive and MtP5CS2 induced by abscisic acid. Under salinity, MtP5CS3 promoter was more active than those of MtP5CS1 and MtP5CS2, as shown by GUS fusions. Translationally fused MtP5CS1-GFP was localized in the cytoplasm, whereas significant proportions of MtP5CS2-GFP and MtP5CS3-GFP were co-localized with rubisco small subunit protein-fused RFP in transformed hairy root cells. Under salinity, RNA silencing of MtP5CS1 or MtP5CS2 strongly induced MtP5CS3 expression, while that of MtP5CS3 decreased free proline content and nodule number. Consistently, Mtp5cs3, a loss-of-function mutant, accumulated much less proline, formed fewer nodules, and fixed nitrogen significantly less efficiently than the wild type under salinity. Thus, MtP5CS3 plays a critical role in regulating stress-induced proline accumulation during symbiotic nitrogen fixation.     

A quick and efficient approach for gene silencing by using triple putative microRNA-based short hairpin RNAs

The RNA interference (RNAi) technique has been widely used in gene function studies. It is typical to screen for effective siRNAs by knocking down targeted genes since a single gene can be suppressed by several siRNAs to varying degrees. The miRNA-based short hairpin RNA (shRNA) is a natural inducer of RNAi and has been used in siRNA expression strategies. We investigated the potential application of multiple putative microRNA-based shRNAs for gene silencing and studied the inhibition efficiency of exogenous GFP and firefly luciferase (luc) by triple human mir155-based shRNA expression vectors. A total of three candidate siRNA sequences targeted against GFP or luc were selected based on an online prediction program. Single and triple miRNA-155-based shRNAs targeted against GFP or luc were transfected into HEK293 cells mediated by the pcDNA₃ vector with an RNA polymerase II-type CMV (cytomegalovirus) promoter. Comparisons with negative control shRNAs revealed that GFP levels were markedly reduced by the triple miRNA-155-based GFP shRNA by fluorescent microscopy. Consistent results from the dual luciferase assay and real-time quantitative RT-PCR revealed that the triple miRNA-155-based GFP shRNA significantly suppressed GFP expression (P < 0.01), without significant differences from the most effective single miRNA-155-based GFP shRNA (P > 0.05). Results from the dual luciferase assay and real-time quantitative RT-PCR revealed that the triple miRNA-155-based luc shRNA significantly suppressed luc expression as the most effective single miRNA-155-based luc shRNA (P < 0.05). These studies demonstrated the gene silencing efficiency mediated by the triple putative miRNA-155-based shRNAs. This suggested that multiple miRNA-based shRNAs are quick and valuable strategies for gene silencing.     

shRNA Off-Target Effects In Vivo: Impaired Endogenous siRNA Expression and Spermatogenic Defects

RNA interference (RNAi) is widely used to determine the function of genes. We chose this approach to assess the collective function of the highly related reproductive homeobox 3 (Rhox3) gene paralogs. Using a Rhox3 short hairpin (sh) RNA with 100% complementarity to all 8 Rhox3 paralogs, expressed from a CRE-regulated transgene, we successfully knocked down Rhox3 expression in male germ cells in vivo. These Rhox3-shRNA transgenic mice had dramatic defects in spermatogenesis, primarily in spermatocytes and round spermatids. To determine whether this phenotype was caused by reduced Rhox3 expression, we generated mice expressing the Rhox3-shRNA but lacking the intended target of the shRNA—Rhox3. These double-mutant mice had a phenotype indistinguishable from Rhox3-shRNA-expressing mice that was different from mice lacking the Rhox3 paralogs, indicating that the Rhox3 shRNA disrupts spermatogenesis independently of Rhox3. Rhox3-shRNA transgenic mice displayed few alterations in the expression of protein-coding genes, but instead exhibited reduced levels of all endogenous siRNAs we tested. This supported a model in which the Rhox3 shRNA causes spermatogenic defects by sequestering one or more components of the endogenous small RNA biogenesis machinery. Our study serves as a warning for those using shRNA approaches to investigate gene functions in vivo.     

PolⅡ型启动子K14实现组织特异RNAi

RNAi(RNA interference,RNAi)是继基因打靶技术后的一种高效的研究基因功能的方法。细胞学实验和小鼠模型的研究结果表明,PolⅡ型启动子可以实现组织特异的RNA干扰,从而为鉴定基因在特定组织中的功能及作用机理提供了一个强有力的研究方法。为了能将这种方法用于转基因绵羊生产,探讨基因与绵羊毛囊发育的关系及其作用机制等,文章利用PolⅡ型CMV启动子和毛囊组织特异表达的人角蛋白14(K14)基因的启动子驱动eGFP-shRNA融合转录本的生成,从而实现敲低目的基因的表达。体外基因表达沉默效率分析(pEGFP-C1-shRNA和psiCHECK-BMP4双质粒共转染Hela细胞)结果表明,6个干扰序列均能有效地抑制BMP4基因的表达,抑制效率达到60%以上;体内表达沉默分析(只转染pEGFP-K14-shRNA质粒转染小鼠皮肤细胞系JB6-C41)的实验结果与体外分析结果相似,除3#序列外,其余干扰序列对BMP4基因的抑制效率都在60%以上,其中5#序列的效率达到80%以上。siRNA诱导的目标基因沉默中mRNA和蛋白水平的下降显著正相关。结果表明,设计构建的由PolⅡ型启动子K14驱动eGFP-shRNA融合转录本的形成,从而实现RNAi的研究方法是可行的,利用这种方法可以实现在特定细胞中敲低目的基因的表达水平。为在大家畜特别是绵羊中应用RNAi的方法分析目的基因在毛囊发育、对不同类型毛囊生长发育的诱导和调节等作用机理的研究提供一个参考方法。     

First Knockdown Gene Expression in Bat (Hipposideros armiger) Brain Mediated by Lentivirus

Lentivirus-mediated RNA interference (RNAi) is a potent experimental tool for investigating gene functions in vitro and in vivo. It has advantages that transgenic technology lacks. However, in vivo applications are difficult to apply in the central nervous system of non-model organisms due to the lack of a standard brain atlas and genetic information. Here, we report the development of an in vivo gene delivery system used in bat brain tissue for the first time, based on lentivirus (LV) vectors expressing short hairpin RNA (shRNA) targeting Hipposideros armiger forkhead box P2 (FoxP2). In vitro transfection into HEK 293T cell with the vector bearing the cassettes encoding FoxP2 shRNA verified the knockdown efficiency. Pseudovirus particles were administered via stereotactic intracerebral microinjection into the anterior cingulate cortex of H. armiger. FoxP2 is of major interest because of its role in sensorimotor coordination and probably in echolocation. Subsequent in situ hybridization validated the in vivo silencing of the target gene. This report demonstrates that LV-mediated expression of RNAi could achieve effective gene silencing in bats, a non-model organism, and will assist in elucidating the functions of bat genes.     

Human U6 promoter drives stronger shRNA activity than its schistosome orthologue in Schistosoma mansoni and human fibrosarcoma cells

Blood flukes or schistosomes are the causative agents of human schistosomiasis, one of the major neglected tropical diseases. Draft genome sequences have been reported for schistosomes, but functional genomics tools are needed to investigate the role and essentiality of the newly reported genes. Vector based RNA interference can contribute to functional genomics analysis for schistosomes. Using mRNA encoding reporter firefly luciferase as a model target, we compared the performance of a schistosome and a human promoter from the U6 gene in driving shRNA in human fibrosarcoma cells and in cultured schistosomes. Further, both a retroviral [Murine leukemia virus (MLV)] and plasmid (piggyBac, pXL-Bac II) vector were utilized. The schistosome U6 gene promoter was 270 bp in length, the human U6 gene promoter was 264 bp; they shared 41% identity. Following transduction of both HT1080 fibrosarcoma cells and schistosomules of Schistosoma mansoni with pseudotyped MLV virions, stronger knockdown of luciferase activity was seen with the virions encoding the human U6 promoter driven shRNA than the schistosome U6 promoter. A similar trend was seen after transfection of HT1080 cells and schistosomules with the pXL-Bac-II constructs-stronger knockdown of luciferase activity was seen with constructs encoding the human compared to schistosome U6 promoter. The findings indicate that a human U6 gene promoter drives stronger shRNA activity than its schistosome orthologue, not only in a human cancer cell line but also in larval schistosomes. This RNA polymerase III promoter represents a potentially valuable component for vector based RNA interference studies in schistosomes and related platyhelminth parasites.     

An inducible system for expression and validation of the specificity of short hairpin RNA in mammalian cells

RNA interference (RNAi) by means of short hairpin RNA (shRNA) has developed into a powerful tool for loss-of-function analysis in mammalian cells. The principal problem in RNAi experiments is off-target effects, and the most vigorous demonstration of the specificity of shRNA is the rescue of the RNAi effects with a shRNA-resistant target gene. This presents its own problems, including the unpredictable relative expression of shRNA and rescue cDNA in individual cells, and the difficulty in generating stable cell lines. In this report, we evaluated the plausibility of combining the expression of shRNA and rescue cDNA in the same vector. In addition to facilitate the validation of shRNA specificity, this system also considerably simplifies the generation of shRNA-expressing cell lines. Since the compensatory cDNA is under the control of an inducible promoter, stable shRNA-expressing cells can be generated before the knockdown phenotypes are studied by conditionally turning off the rescue protein. Conversely, the rescue protein can be activated after the endogenous protein is completely repressed. This approach is particularly suitable when prolonged expression of either the shRNA or the compensatory cDNA is detrimental to cell growth. This system allows a convenient one-step validation of shRNA and generation of stable shRNA-expressing cells.