RNAi技术在转基因动物中的应用

RNAi可以作为一种有效的工具用来产生转录后沉默的效果,从而抑制特定基因的表达,已经在线虫、果蝇、小鼠、大鼠等模式生物中得到成功应用。RNAi转基因小鼠的出现,使得在哺乳动物整体水平研究靶基因的敲低成为可能。文章以RNAi转基因小鼠为代表,就转基因载体的设计策略、基因敲除与基因敲低的比较、RNAi转基因动物的优势以及目前存在的缺陷等作一总结,并展望了RNAi转基因动物对功能基因组研究的贡献以及应用前景。

     

A rapid and scalable system for studying gene function in mice using conditional RNA interference

RNA interference is a powerful tool for studying gene function, however, the reproducible generation of RNAi transgenic mice remains a significant limitation. By combining optimized fluorescence-coupled miR30-based shRNAs with high efficiency ES cell targeting, we developed a fast, scalable pipeline for the production of shRNA transgenic mice. Using this system, we generated eight tet-regulated shRNA transgenic lines targeting Firefly and Renilla luciferases, Oct4 and tumor suppressors p53, p16(INK4a), p19(ARF) and APC and demonstrate potent gene silencing and GFP-tracked knockdown in a broad range of tissues in?vivo. Further, using an shRNA targeting APC, we illustrate how this approach can identify predicted phenotypes and also unknown functions for a well-studied gene. In addition, through regulated gene silencing we validate APC/Wnt and p19(ARF) as potential therapeutic targets in T?cell acute lymphoblastic leukemia/lymphoma and lung adenocarcinoma, respectively. This system provides a cost-effective and scalable platform for the production of RNAi transgenic mice targeting any mammalian gene. PAPERCLIP:     

A pipeline for the generation of shRNA transgenic mice

RNA interference (RNAi) is an extremely effective tool for studying gene function in almost all metazoan and eukaryotic model systems. RNAi in mice, through the expression of short hairpin RNAs (shRNAs), offers something not easily achieved with traditional genetic approaches-inducible and reversible gene silencing. However, technical variability associated with the production of shRNA transgenic strains has so far limited their widespread use. Here we describe a pipeline for the generation of miR30-based shRNA transgenic mice that enables efficient and consistent targeting of doxycycline-regulated, fluorescence-linked shRNAs to the Col1a1 locus. Notably, the protocol details crucial steps in the design and testing of miR30-based shRNAs to maximize the potential for developing effective transgenic strains. In all, this 14-week procedure provides a fast and cost-effective way for any laboratory to investigate gene function in vivo in the mouse.     

Silencing of the Pink1 gene expression by conditional RNAi does not induce dopaminergic neuron death in mice

Transgenic RNAi, an alternative to the gene knockout approach, can induce hypomorphic phenotypes that resemble those of the gene knockout in mice. Conditional transgenic RNAi is an attractive choice of method for reverse genetics in vivo because it can achieve temporal and spatial silencing of targeted genes. Pol III promoters such as U6 are widely used to drive the expression of RNAi transgenes in animals. Tested in transgenic mice, a Cre-loxP inducible U6 promoter drove the broad expression of an shRNA against the Pink1 gene whose loss-of-functional mutations cause one form of familial Parkinson's disease. The expression of the shRNA was tightly regulated and, when induced, silenced the Pink1 gene product by more than 95% in mouse brain. However, these mice did not develop dopaminergic neurodegeneration, suggesting that silencing of the Pink1 gene expression from embryo in mice is insufficient to cause similar biochemical or morphological changes that are observed in Parkinson's disease. The results demonstrate that silencing of the PINK1 gene does not induce a reliable mouse model for Parkinson's disease, but that technically the inducible U6 promoter is useful for conditional RNAi in vivo.     

RUSH and CRUSH: A rapid and conditional RNA interference method in mice

RNA interference (RNAi) is a powerful approach to phenocopy mutations in many organisms. Gold standard conventional knock‐out mouse technology is labor‐ and time‐intensive; however, off‐target effects may confound transgenic RNAi approaches. Here, we describe a rapid method for conditional and reversible gene silencing in RNAi transgenic mouse models and embryonic stem (ES) cells. RUSH and CRUSH RNAi vectors were designed for reversible or conditional knockdown, respectively, demonstrated using targeted replacement in an engineered ROSA26^lacZ ES cell line and wildtype V6.5 ES cells. RUSH was validated by reversible knockdown of Dnmt1 in vitro. Conditional mouse model production using CRUSH was expedited by deriving ES cell lines from Cre transgenic mouse strains (nestin, cTnnT, and Isl1) and generating all‐ES G₀ transgenic founders by tetraploid complementation. A control CRUSH^GFP RNAi mouse strain showed quantitative knockdown of GFP fluorescence as observed in compound CRUSH^GFP, Ds‐Red Cre‐reporter transgenic mice, and confirmed by Western blotting. The capability to turn RUSH and CRUSH alleles off or on using Cre recombinase enables this method to rapidly address questions of tissue‐specificity and cell autonomy of gene function in development. genesis 52:39–48, 2014. © 2013 Wiley Periodicals, Inc.     

Two novel proteins, MRL7 and its paralog MRL7-L, have essential but functionally distinct roles in chloroplast development and are involved in plastid gene expression regulation in Arabidopsis

Chloroplast development requires the coordinated action of various proteins, many of which remain to be identified. Here, we report two novel genes, Mesophyll-cell RNAi Library line 7 (MRL7) and MRL7-Like (MRL7-L), that are involved in this process. An Arabidopsis knock-down transgenic plant (MRL7-RNAi) with delayed-greening phenotype was isolated from an RNA interference (RNAi) transformant library. Cotyledons and young leaves of MRL7-RNAi were pale in seedlings and gradually greened as the plant matured, while a knock-out in the MRL7 gene was seedling lethal. The MRL7 protein was shown to co-localize with a marker protein for nucleoids in chloroplasts, indicative of a role for the protein in chloroplast nucleic acid metabolism. Accordingly, chloroplast development was arrested upon loss of MRL7 function and the expression of plastid-encoded genes transcribed by plastid-encoded RNA polymerase (PEP) was significantly reduced in MRL7 knock-down and knock-out plants. A paralog of MRL7 (MRL7-L) was identified in the Arabidopsis genome. Both MRL7 and MRL7-L are only found in land plants and encode previously uncharacterized proteins without any known conserved domain. Like MRL7, knock-down of MRL7-L also resulted in a virescent phenotype, and a similar effect on plastid gene expression. However, the MRL7-L protein was localized to the chloroplast stroma. Taken together, our data indicate that the two paralogous proteins MRL7 and MRL7-L have essential but distinct roles during early chloroplast development and are involved in regulation of plastid gene expression.     

A high-throughput gene-silencing system for the functional assessment of defense-related genes in barley epidermal cells

Large-scale gene silencing by RNA interference (RNAi) offers the possibility to address gene function in eukaryotic organisms at a depth unprecedented until recently. Although genome-wide RNAi approaches are being carried out in organisms like Caenorhabditis elegans, Drosophila spp. or human after the corresponding tools had been developed, knock-down of only single or a few genes by RNAi has been reported in plants thus far. Here, we present a method for high-throughput, transient-induced gene silencing (TIGS) by RNAi in barley epidermal cells that is based on biolistic transgene delivery. This method will be useful to address gene function of shoot epidermis resulting in cell-autonomous phenotypes such as resistance or susceptibility to the powdery-mildew fungus Blumeria graminis f. sp. hordei. Gene function in epidermal cell elongation, stomata regulation, or UV resistance might be addressed as well. Libraries of RNAi constructs can be built up by a new, cost-efficient method that combines highly efficient ligation and recombination by the Gateway cloning system. This method allows cloning of any blunt-ended DNA fragment without the need of adaptor sequences. The final RNAi destination vector was found to direct highly efficient RNAi, as reflected by complete knock-down of a cotransformed green fluorescent protein reporter gene as well as by complete phenolcopy of the recessive loss-of-function mlo resistance gene. By using this method, a role of the t-SNARE protein HvSNAP34 in three types of durable, race-nonspecific resistance was observed.     

Expression of RNA-interference/antisense transgenes by the cognate promoters of target genes is a better gene-silencing strategy to study gene functions in rice

Antisense and RNA interference (RNAi)-mediated gene silencing systems are powerful reverse genetic methods for studying gene function. Most RNAi and antisense experiments used constitutive promoters to drive the expression of RNAi/antisense transgenes; however, several reports showed that constitutive promoters were not expressed in all cell types in cereal plants, suggesting that the constitutive promoter systems are not effective for silencing gene expression in certain tissues/organs. To develop an alternative method that complements the constitutive promoter systems, we constructed RNAi and/or antisense transgenes for four rice genes using a constitutive promoter or a cognate promoter of a selected rice target gene and generated many independent transgenic lines. Genetic, molecular, and phenotypic analyses of these RNAi/antisense transgenic rice plants, in comparison to previously-reported transgenic lines that silenced similar genes, revealed that expression of the cognate promoter-driven RNAi/antisense transgenes resulted in novel growth/developmental defects that were not observed in transgenic lines expressing constitutive promoter-driven gene-silencing transgenes of the same target genes. Our results strongly suggested that expression of RNAi/antisense transgenes by cognate promoters of target genes is a better gene-silencing approach to discovery gene function in rice.     

Conditional RNAi in mice

RNA interference (RNAi)-mediated gene knockdown has developed into a routine method to assess gene function in cultured mammalian cells in a fast and easy manner. For the use of RNAi in mice, short hairpin (sh) RNAs expressed stably from the genome are a fast alternative to conventional knockout approaches. We developed a strategy for complete or conditional gene knockdown in mice, where the Cre/loxP system is used to activate RNAi in a time and tissue dependent manner. Alternatively doxycycline controlled shRNA expression vectors can be used for conditional gene silencing. Single copy RNAi constructs are placed into the Rosa26 locus of ES cells by recombinase mediated cassette exchange and transmitted through the germline of chimeric mice. The shRNA transgenic offspring can be either directly used for phenotypic analysis or are further crossed to a Cre transgenic strain to activate conditional shRNA vectors. The site specific insertion of single copy shRNA vectors allows the expedite and reproducible production of knockdown mice and provides an easy and fast approach to assess gene function in vivo.     

猪Nanog基因四环素诱导干扰载体的构建和鉴定

Nanog基因是在早期胚胎和干细胞等多能性细胞中特异表达的重要基因,但有关猪Nanog基因功能的相关研究甚少。四环素诱导干扰载体是一种可通过四环素等药物条件性诱导干扰目的基因的载体,尤其适用于在发育过程中起着关键作用的基因沉默。常规的四环素干扰系统为二元载体,与一元载体相比获得针对特定基因干扰的稳定细胞系所需周期更长。首先通过构建pGenesil 1.0-shRNA重组干扰载体,瞬时转染稳定过表达猪Nanog基因的猪胎儿成纤维细胞后通过Realtime-PCR筛选出干扰效率可达80%以上的干扰片段。之后将筛选得到的干扰片段插入到改造的一元四环素诱导干扰载体TREsilencer,对稳定表达猪Nanog基因的猪胎儿成纤维细胞进行了瞬时转染。实验分别通过光密度检测以及Realtime-PCR检测了不同浓度doxycycline的诱导效率和干扰效率。结果表明,所构建的四环素诱导干扰载体TREsilencer-shRNA5随着四环素浓度的增加,诱导Nanog基因的干扰效率增加,在处理浓度为1μg/ml时干扰效率可达70%以上,为后续得到可诱导的稳定干扰猪Nanog基因的细胞系和进一步研究猪Nanog基因功能奠定了基础。     

Efficient delivery of RNA Interference to peripheral neurons in vivo using herpes simplex virus

Considerable interest has been focused on inducing RNA interference (RNAi) in neurons to study gene function and identify new targets for disease intervention. Although small interfering RNAs (siRNAs) have been used to silence genes in neurons, in vivo delivery of RNAi remains a major challenge limiting its applications. We have developed a highly efficient method for in vivo gene silencing in dorsal root ganglia (DRG) using replication-defective herpes simplex viral (HSV-1) vectors. HSV-mediated delivery of short-hairpin RNA (shRNA) targeting reporter genes resulted in highly effective and specific silencing in neuronal and non-neuronal cells in culture and in the DRG of mice in vivo including in a transgenic mouse model. We further establish proof of concept by demonstrating in vivo silencing of the endogenous trpv1 gene. These data are the first to show silencing in DRG neurons in vivo by vector-mediated delivery of shRNA. Our results support the utility of HSV vectors for gene silencing in peripheral neurons and the potential application of this technology to the study of nociceptive processes and in pain gene target validation studies.     

昆虫RNAi技术及其应用

RNAi是近几年发展起来的抑制基因表达的新技术。部分昆虫存在RNAi信号的系统性传播现象,可以将dsRNA直接注射进昆虫的卵、血腔或局部组织,引发远距离靶基因的特异性沉默,建立起了Embryo RNAi,Larval RNAi,Adult RNAi,Parental RNAi,Feeding RNAi和基于转基因技术的可遗传RNAi等昆虫RNAi技术,使RNAi迅速成为了研究昆虫尤其是非模式昆虫基因功能的主要方法。文章拟就RNAi的系统性、昆虫RNAi技术及其应用进行综述。     

Small interfering RNA and gene silencing in transgenic mice and rats

After short duplexes of synthetic 21-23 nt RNAs (siRNA) were reported to be effective in silencing specific genes, a vector-based approach for siRNAs was demonstrated in mammalian cultured cell lines. However, the effect of RNA interference (RNAi) on various differentiated cells in live animals remains unknown. In this report, we demonstrate that transgenically supplied siRNA can silence ubiquitously expressed enhanced green fluorescent protein in every part of the mouse and rat body. These results suggest that transgenic RNAi could function as an alternative method of gene silencing by applying homologous recombination to embryonic stem (ES) cells, and should be successful even in species where ES cell lines remain unestablished.     

基因敲除鼠疾病模型的研究进展

基因敲除是研究生物体基因功能的有效手段。通过基因敲除建立的鼠疾病模型,在研究基因功能及人类疑难病症致病机制等方面发挥着前所未有的作用。本文对目前已获得的基因敲除鼠疾病模型进行了分类和总结,为相关研究的展开奠定了基础。 Abstract:The knock-out technology is an effective means in studying the gene function of organism.The disease model of gene knock-out mouse is of significance in understanding the gene function and pathogenesis of human disease.The available models of gene knock-out mouse are classified and summarized to promote the development of related research.