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Design of artificial small regulatory trans-RNA for gene knockdown in Bacillus subtilis

Synthetic and Systems Biotechnology 2023年第1期8卷 61-68页

作者： Guobin Yin Anqi Peng Luyao Zhang Yang Wang Guocheng Du Jian Chen Zhen Kang The Science Center for Future Foods Jiangnan UniversityWuxi214122China The Key Laboratory of Carbohydrate Chemistry and Biotechnology Ministry of EducationSchool of BiotechnologyJiangnan UniversityWuxi214122China The Key Laboratory of Industrial Biotechnology Ministry of EducationSchool of BiotechnologyJiangnan UniversityWuxi214122China

Bacillus subtilis as the Gram-positive model bacterium has been widely used in synthetic biology and biotechnology while the regulatory RNA tools for B.subtilis are still not fully explored.Here,a bottom-up approach is proposed for designing artificial trans-acting sRNAs.By engineering the intrinsic sRNA SR6,a minimized core scaffold structure consisting of an 8 bp stem,a 4 nt loop,and a 9 nt polyU tail was generated and proven to be sufficient for constructing sRNAs with strong repression activity(83%).Moreover,we demonstrate this artificial sRNA system functions well in an hfq-independent manner and also achieves strong repression efficiency in Escherichia coli(above 80%).A structure-based sRNA design principle was further developed for the automatic generation of custom sRNAs with this core scaffold but various sequences,which facilitates the manipulation and avoids structure disruption when fusing any base-pairing sequence.By applying these auto-designed sRNAs,we rapidly modified the cell morphology and biofilm formation,and regulated metabolic flux toward acetoin biosynthesis.This sRNA system with cross-species regulatory activities not only enriched the gene regulation toolkit in synthetic biology for B.subtilis and E.coli but also enhanced our understanding of trans-acting sRNAs.

关键词： gene knockdown Posttranscriptional regulation Small noncoding RNA Synthetic biology Cross-species Bacillus subtilis

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Let-7a gene knockdown protects against cerebral ischemia/reperfusion injury

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Neural Regeneration Research 2016年第2期11卷 262-269页

作者： Zhong-kun Wang Fang-fang Liu Yu Wang Xin-mei Jiang Xue-fan Yu Department of Neurology First Hospital Jilin University Department of Neurology Jilin Central Hospital Department of Hepatopancreatobiliary Surgery the Second Hospital of Jilin University

The micro RNA（mi RNA） let-7 was one of the first mi RNAs to be discovered, and is highly conserved and widely expressed among species. let-7 expression increases in brain tissue after cerebral ischemia/reperfusion injury; however, no studies have reported let-7 effects on nerve injury after cerebral ischemia/reperfusion injury. To investigate the effects of let-7 gene knockdown on cerebral ischemia/reperfusion injury, we established a rat model of cerebral ischemia/reperfusion injury. Quantitative reverse transcription-polymerase chain reaction demonstrated that 12 hours after cerebral ischemia/reperfusion injury, let-7 expression was up-regulated, peaked at 24 hours, and was still higher than that in control rats after 72 hours. Let-7 gene knockdown in rats suppressed microglial activation and inflammatory factor release, reduced neuronal apoptosis and infarct volume in brain tissue after cerebral ischemia/reperfusion injury. Western blot assays and luciferase assays revealed that mitogen-activated protein kinase phosphatase-1（MKP1） is a direct target of let-7. Let-7 enhanced phosphorylated p38 mitogen-activated protein kinase（MAPK） and c-Jun N-terminal kinase（JNK） expression by down-regulating MKP1. These findings suggest that knockdown of let-7 inhibited the activation of p38 MAPK and JNK signaling pathways by up-regulating MKP1 expression, reduced apoptosis and the inflammatory reaction, and exerted a neuroprotective effect following cerebral ischemia/reperfusion injury.

关键词： nerve regeneration cerebral ischemia/reperfusion injury let-7 mitogen-activated protein kinase phosphatase-1 apoptosis microglia inflammation mitogen-activated protein kinase neurons c-Jun N-terminal kinase gene knockdown brain injury neural regeneration

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Impact of Pitx3 gene knockdown on glial cell line-derived neurotrophic factor transcriptional activity in dopaminergic neurons

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Neural Regeneration Research 2017年第8期12卷 1347-1351页

作者： Jing Chen Xiao-yu Kang Chuan-xi Tang Dian-shuai Gao Experiment Teaching Center of Morphology Xuzhou Medical University Xuzhou Jiangsu Province China Teaching and Research Section of Neurobiology and Anatomy Xuzhou Medical University Xuzhou Jiangsu Province China

Pitx3 is strongly associated with the phenotype, differentiation, and survival of dopaminergic neurons. The relationship between Pitx3 and glial cell line-derived neurotrophic factor（GDNF） in dopaminergic neurons remains poorly understood. The present investigation sought to construct and screen a lentivirus expression plasmid carrying a rat Pitx3 short hairpin（sh）RNA and to assess the impact of Pitx3 gene knockdown on GDNF transcriptional activity in MES23.5 dopaminergic neurons. Three pairs of interference sequences were designed and separately ligated into GV102 expression vectors. These recombinant plasmids were transfected into MES23.5 cells and western blot assays were performed to detect Pitx3 protein expression. Finally, the most effective Pitx3 sh RNA and a dual-luciferase reporter gene plasmid carrying the GDNF promoter region（GDNF-luciferase） were cotransfected into MES23.5 cells. Sequencing showed that the synthesized sequences were identical to the three Pitx3 interference sequences. Inverted fluorescence microscopy revealed that the lentivirus expression plasmids carrying Pitx3-sh RNA had 40-50% transfection efficiency. Western blot assay confirmed that the corresponding Pitx3 of the third knockdown sequence had the lowest expression level. Dual-luciferase reporter gene results showed that the GDNF transcriptional activity in dopaminergic cells cotransfected with both plasmids was decreased compared with those transfected with GDNF-luciferase alone. Together, the results showed that the designed Pitx3-sh RNA interference sequence decreased Pitx3 protein expression, which decreased GDNF transcriptional activity.

关键词： nerve regeneration neurodegeneration Parkinson＇s disease glial cell line-derived neurotrophic .factor Pitx3 MES23.5 cells shorthairpin RNA gene knockdown plasmid dual-luciferase reporter gene neural regeneration

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Investigation of TbfA in Riemerella anatipestifer using plasmid-based methods for gene over-expression and knockdown

Investigation of TbfA in Riemerella anatipestifer using plas...

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中国畜牧兽医学会动物微生态学分会第五届第十二次全国学术...

作者： MaFeng Liu MengYi Wang DeKang Zhu MingShu Wang RenYong Jia Shun Chen KunFeng Sun Qiao Yang Ying Wu XiaoYue Chen Francis Biville AnChun Cheng Institute of Preventive Veterinary Medicine Sichuan Agricultural University Avian Disease Research Center College of Veterinary Medicine of Sichuan Agricultural University Key Laboratory of Animal Disease and Human Health of Sichuan Province Sichuan Agricultural University Unite des Infections Bacteriennes Invasives Departement Infection et EpidemiologieInstitut Pasteur

Riemerella anatipestifer is a duck pathogen that has caused serious economic losses to the duck industry worldwide.Despite this,there are few reported studies of the physiological and pathogenic mechanisms of Riemerella anatipestifer infection.In previous study,we have shown that TonB1 and TonB2 were involved in hemin uptake.TonB family protein(TbfA) was not investigated,since it was failed to knockout at that time.Here,we used a plasmid based gene overexpression and knockdown to investigate it.First,we constructed three Escherichia-Riemerella anatipestifer shuttle vectors containing three different native Riemerella anatipestifer promoters.The shuttle plasmids were introduced into Riemerella anatipestifer ATCC11845 by conjugation at an efficiency of 5×10 antibiotic-resistant transconjugants per recipient cell.Based on the high-expression shuttle vector pLMF03,a method for gene knockdown was established.knockdown of TbfA in Riemerella anatipestifer ATCC11845 decreased the organism’s growth ability in TSB medium but did not affect its hemin utilization.In contrast,over-expression of TbfA in Riemerella anatipestifer ATCC11845△tonB1△tonB2 significantly promoted the organism’s growth in TSB medium but significantly inhibited its hemin utilization.Collectively,these findings suggest that TbfA is not involved in hemin utilization by Riemerella anatipestifer.

关键词： Riemerella anatipestifer TonB family protein（TbfA） gene overexpression gene knockdown Investigate

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The emerging potential of siRNA nanotherapeutics in treatment of arthritis

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Asian Journal of Pharmaceutical Sciences 2023年第5期18卷 3-25页

作者： Anjali Kumari Amanpreet Kaur Geeta Aggarwal Centre for Advanced Formulation Technology Delhi Pharmaceutical Sciences and ResearchNew Delhi 110017India School of Pharmaceutical Sciences Delhi Pharmaceutical Sciences and Research UniversityNew Delhi 110017India

RNA interference(RNAi)using small interfering RNA(siRNA)has shown potential as a therapeutic option for the treatment of arthritis by silencing specific genes.However,siRNA delivery faces several challenges,including stability,targeting,off-target effects,endosomal escape,immune response activation,intravascular degradation,and renal clearance.A variety of nanotherapeutics like lipidic nanoparticles,liposomes,polymeric nanoparticles,and solid lipid nanoparticles have been developed to improve siRNA cellular uptake,protect it from degradation,and enhance its therapeutic efficacy.Researchers are also investigating chemical modifications and bioconjugation to reduce its immunogenicity.This review discusses the potential of siRNA nanotherapeutics as a therapeutic option for various immune-mediated diseases,including rheumatoid arthritis,osteoarthritis,etc.siRNA nanotherapeutics have shown an upsurge of interest and the future looks promising for such interdisciplinary approach-based modalities that combine the principles of molecular biology,nanotechnology,and formulation sciences.

关键词： RNAi siRNA Nanotherapeutics gene knockdown Osteoarthritis Rheumatoid arthritis

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Effects of SHANK2 gene knockdown on early neural development of induced pluripotent stem cell derived neuron

Effects of SHANK2 gene knockdown on early neural development...

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中国神经科学学会第十二届全国学术会议

作者： Chen Xiaoxia Huang Zhonghai Kang Sai Yu Panpan Shi Lingling Guangdong-Hongkong-Macau Institute of CNS Regeneration Joint International Research Laboratory of CNS Regeneration Ministry of Education of PRC Jinan University Co-innovation Center of Neuroregeneration Nantong University

OBJECTIVE: To investigate the effect of SHANK2 knockdown on the early morphological development of neurons derived from human induced pluripotent stem cell（iPSCs）. METHODS: Induced pluripotent stem cells（iPSCs） were differentiated into neural progenitor cells（NPC） and NPCs were differentiated into neurons. The models were divided into sh Control group（infected with control Lenti virus packed with a red fluorescent protein expression sequence） and sh SHANK2 group（infected with SHANK2 Lenti virus packed with a red fluorescent protein expression sequence）. The soma area, growth cone area, neurite length and branches number of neurons were analyzed by immunohistochemical method on selected differentiation days（Day3, Day6, Day9, Day12, and Day15）. RESULTS:（1） The model with NPC was generated in vitro significantly.（2） Compared with control, SHANK2 gene knockdown resulted in the decrease of soma area at Day9、Day12、Day15.（3） Compared with control, the number of total branches, primary branches, and primary branches with SHANK2 gene knockdown were reduced, and the length of the neurite, axon, and dendrite were reduced at Day6、Day9、Day12、Day15.（4） Compared with control, SHANK2 gene knockdown resulted in the decrease of three subtypes of growth cone area bluntended（collapsed with no visible filopodia or lamellipodia）, filopodial（growth cones with numerous filopodia and a small or absent lamellipodial veil）, or lamellipodial（well spread growth cones with elaborate lamellipodia） at Day6 、 Day9 、 Day12 、 Day15. CONCLUSION: The early development of neurons were affected by SHANK2 gene knockdown, leading to abnormal neuronal morphology development.

关键词： SHANK2 gene Induced pluripotent stem cells gene knockdown Neuronal morphology

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Improved neurite outgrowth on central nervous system myelin substrate by siRNA-mediated knockdown of Nogo receptor

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Chinese Journal of Traumatology 2016年第1期19卷 16-24页

作者： Sheng-Hao Ding Ying-Hui Bao jian-Hong Shen Guo-Yi Gao Yao-Hua Pan Qi-Zhong Luo Ji-Yao Jiang Department ofNeurosurgery RenJi Hospital School of Medicine ShanghaiJiao Tong University Shanghai 200127 China Department of Neurosurgery Affiliated Hospital of Nantong University Nantong 226001 China

OBJECTIVE:To investigate the in vitro effect of short interfering RNAs (siRNAs) against Nogo receptor (NgR) on neurite outgrowth under an inhibitory substrate of central nervous system (CNS) myelin.;METHODS:Three siRNA sequences against NgR were designed and transfected into cerebellar granule cells (CGCs) to screen for the most effcient sequence of NgR siRNA by using reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence staining. NgR siRNA sequence 1 was found the most efficient which was then transfected into the CGCs grown on CNS myelin substrate to observe its disinhibition for neurite outgrowth.;RESULTS:Compared with the scrambled control sequence of siRNA, the NgR siRNA sequence 1 significantly decreased NgR mRNA level at 24 h and 48 h (p knockdown of NgR expression contributes to neurite outgrowth in vitro.

关键词： RNA interference Neurite outgrowth Nogo receptor gene knockdown

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RNAi Mediated Silencing of gene Encoding 1-Deoxy-D-Xylulose-5-Phosphate Reductoisomerase (DXR) in <i>Centella asiatica</i>

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American Journal of Plant Sciences 2020年第11期11卷 1723-1738页

作者： Richa Sharma Ratna Kalita Basanta Kr. Borah Mahendra K. Modi Priyabrata Sen Department of Agricultural Biotechnology Assam Agricultural University Jorhat India

Centella asiatica (L.) is one of the most valuable medicinal plants since prehistoric times. The pharmaceutical importance of this herb is due to the accumulation of large quantities of pentacyclic triterpenoid saponins, collectively known as centelloids synthesized by the isoprenoid biosynthesis pathway. Biosynthesis of triterpenoid in the plants proceeds via either of the two pathways, viz. Mevalonate (MVA) pathway (in the cytosol) or 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (in plastid). In Centella, the pathway leading to the accumulation of triterpenoid is still not known or elucidated. Thus, to know whether the MVA or MEP pathway or a cross-talk between the pathway leads to the biosynthesis of triterpenoid, silencing the key regulatory gene using RNAi tool, of each of the pathway and then analyze a metabolite is an efficient approach. The key regulatory enzyme of the MVA pathway i.e. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) has already been successfully silenced using RNAi tool [1]. In the present study, the 1-deoxy-D-xylulose-5-

关键词： Centella DXR RNAi gene knockdown Secondary Metabolite Agro Infiltration

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Identification of the role of phosphodiesterase in mediating memory:implication in Alzheimer’s disease

Identification of the role of phosphodiesterase in mediating...

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中国神经科学学会第九届全国学术会议暨第五次会员代表大会

作者： Rolf HANSEN Marco CONTI Steven P.WILSON James M.O'DONNELL Departments of Behavioral Medicine & Psychiatry and Physiology & Pharmacology West Virginia University Health Sciences CenterMorgantownWV26506USA Division of Reproductive Biology Department of Obstetrics & GynecologyUniversity of CaliforniaSan FranciscoCA 94143USA Department of Pharmacology Physiologyand NeuroscienceUniversity of South Carolina School of MedicineColumbiaSC 29208USA

＜正＞Objective Phosphodiseterase（PDE） is a superfamily of enzymes that catalyzes the hydrolysis of intracellular cAMP and/or cGMP.Among the 11 PDE families（PDE1-11）,PDE4 is the most important family in the control of intracellular cAMP signaling.It has been well established that PDE4 plays an essentia]role in the mediation of memory.However,it is not known whether PDE4 is involved in memory associated with Alzheimer’s disease（AD）. The objective of this study was to identify the role of PDE4 and its isoforms（PDE4A-D） in AD-related memory. Methods Mice deficient in PDE4A,PDE4B,or PDE4D were microinfused withβ-amyloid peptide 1-42（Aβ42;0.4μg/side） or vehicle into bilateral dorsal hippocampi before memory tests using the object recognition,passive avoidance,and Morris water-maze tasks.gene silencing was used to identify the role of PDE4 variants in memory. Expression of phosphorylated CREB（pCREB） in the hippocampus was also examined using Western blotting analysis. Results Mice deficient in PDE4A or PDE4D,but not PDE4B,displayed memory enhancement in one or all of the memory tests.PDE4A or PDE4D knockout mice also exhibited reversal of Aβ42-induced memory impairment, which was observed in wild-type mice in the object recognition,passive avoidance,and water-maze tests.Further, microinfusions of lentiviral vectors containing microRNAs（miRNAs） that target long-form PDE4D splice variants mimicked the ability of PDE4D deficiency to enhance memory in normal mice;the miRNAs also reversed Aβ42- induced memory deficits in the radial arm maze test in rats.Finally,Aβ42 decreased pCREB levels in the hippocampus of wild-type mice or negative control sequence-treated rats,but not mice deficient in PDE4A or PDE4D nor rats treated with PDE4D-miRNAs.Conclusion These results suggest that both PDE4A and PDE4D,in particular the long-form PDE4D isoforms,play an important role in the mediation of memory associated with Alzheimer’s disease. Targeting PDE4A or PDE4D may be a novel approach to treatment of disorders affecting memory and cognit

关键词： phosphodiesterase memory Alzheimer’s disease gene knockout gene knockdown PDE4 subtypes CREB miRNAs

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Detection on the function of CD4+T cells in micro RNA-126 knockdown mice

Detection on the function of CD4+T cells in micro RNA-126 kn...

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第十届全国免疫学学术大会

作者： Panpan Cui Yan Hu Yijing Tao Chao Chen Juanjuan Zhao Mengmeng Guo Ya Zhou Lin Xu Zunyi Medical College

Objective:To investigate the change on function of CD4+T cells in micro RNA-126 knockdown(KD)mice and explore its significance.Methods:The expression level of mature mi RNA-126 in CD4+CD62L+T cells purified by MACS was analyzed by specific probe of real-time PCR assay.Furthermore,the expression of CD69,CD62L and CD44 molecules,as well as intracellular proliferating nuclear antigen Ki-67,in CD4+T cells were detected by FACS.Moreover,the apoptosis of CD4+T cells was analyzed by Annexin V/PI staining assay.Finally,the m RNA relative expression of CD4+T cells function related cytokines were determined by Real-time PCR assay.Results:Compared with those in WT mice,the relative expression level of mature mi RNA-126 in CD4+T cells was dramatically decreased in mi R-126 KD mice(P<0.05).FACS results further showed that the proportion of CD62L in CD4+T cells was significantly decreased,while the proportion of CD69,CD44 and Ki-67 were remarkably increased(P<0.05).Meanwhile,the apoptosis of CD4+T cells in vivo was dramatically decreased in mi R-126 KD mice(P<0.05).Finally,Real-time PCR results indicated that the m RNA expression of IL-4 and IL-10 in CD4+T cells were significantly downregulated,but that of IL-12,TGF-b,TNF-a and IFN-g were obviously up-regulated(P<0.05).Conclusion:mi R-126 KD could significantly affect the functional ability of CD4+T cell in vivo,which providing the preliminarily experimental basis for the study on the mechanism of mi RNA-126 regulation in the function of CD4+T cells.

关键词： MicroRNA-126 gene knockdown CD4+T cells Proliferation Cytokine

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