ONX-0914_PR-957_Proteases_Proteasome_CAS号960374-59-8说明书_AbMole中国

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CelLyticTM系列蛋白裂解液

CelLyticTM系列蛋白裂解液
高效率操作步骤简单,省时高得率得率远优于传统冻融或超声法高活性温和非变性条件下抽提活性蛋白高兼容与蛋白酶抑制剂、鳌合剂、离液剂等很好兼容抽提的蛋白无需去除CelLyticTM 试剂即可进行下游实验：亲和纯化 Western blot 凝胶迁移检测报告基因检测……
566.28 2136.42 566.28 3758.04 2535.39 3938.22
501.93 3320.46
566.28 2084.94 3989.7
527.67 3629.34 5302.44
促销价¥
384.81 2375.80 685.97 2375.80 409.91 1505.79 2651.86 1054.05 1648.00 393.18 1187.90 217.50 711.07 4341.69 368.08 1271.56
哺乳动物细胞和组织裂解
C2978
CelLytic™ M Cell Lysis Reagent
C3228
CelLytic™ MT Cell Lysis Reagent
CE0500 NXTRACT
R0278
CelLytic™ MEM Protein Extraction Kit CelLytic™ NuCLEAR™ Extraction Kit For mammalian tissue or cultured cells RIPA Buffer
P8215
Protease Inhibitor Cocktail for use with fungal and yeast extracts
P8465
Protease Inhibitor Cocktail for use with bacterial cell extracts

为何美国食品和药物管理局推荐肽核酸荧光原位杂交作为快速检测脓毒症致病菌的技术？

２卷第４期
ＪＴａｍｕｇ２１ｌ１．ｒａｓｒ，ｏ０１２ｕ．４
白尿和高血钾等，要时做血液透析。受压过久、需移除重物前未束扎肢体近端、未及时彻底切开减压，都容易促成其发生。伤员尽早被解救并及时全面治疗，是预防和减少综合征和肾损害的关键Ｊ则。本组９例筋膜室切开后仍有ＳＲＩＳ表现，肢肿胀不患减，示病灶处理不彻底；经３～提７次清创，死肌坏肉完全切除后渐愈。通过本组最重的３例，者体笔会，一些看似唯截肢才能挽救生命的伤员，复彻底反
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。
［］徐家相．３地震灾害的紧急医疗救援［］中华急诊医学Ｊ．
杂志，０，４７：５５８２５１（）５７— ５．０
［］王正国．４灾难和事故的创伤救治［．Ｍ］北京：民卫生人
出版社，０５３６～８．２０：３３５
２０２２）７０８，６（：５１～５５９．
（收稿日期：２１Ｏ —２；修回日期：２１ｏ —３）００一１５００一４０
（文编辑：贺本羽）
未处理骨折，仅将 “ 开放伤变闭合伤 ” 这在气性坏，
・
临床问答・
但如无局部和全身适合条件，则该菌即使
存在也不一定致病ｊ。减少组织缺血、消除坏死病灶和避免厌氧环境是防止气性坏疽的关键。具体措

Notch4_mRNA在口咽鳞癌组织中表达变化及其敲降癌细胞株增殖迁移侵袭能力观察

Notch4 mRNA 在口咽鳞癌组织中表达变化及其敲降癌细胞株增殖迁移侵袭能力观察薛绪亭1，张宇良1，郑希望1，董振1，2，刘红亮1，张春明1，21 山西医科大学第一医院耳鼻咽喉头颈肿瘤山西省重点实验室，太原 030001；2 山西医科大学第一医院耳鼻咽喉头颈外科摘要：目的　观察Notch4 mRNA 在口咽鳞癌组织和细胞株中的表达变化及敲降Notch4 mRNA 表达的口咽鳞癌细胞株增殖、迁移和侵袭能力。

方法　收集12对口咽鳞癌组织与癌旁组织，采用RT -qPCR 法检测癌组织、癌旁组织中Notch4 mRNA 。

培养口咽鳞癌细胞株Detroit 562、WSU -HN -30及对照细胞株HaCaT 、2BS ，检测细胞株中Notch4 mRNA 。

将人口咽鳞癌细胞株Detroit 562和WSU -HN -30随机分为si -NC 组、si -Notch4-1组和si -Notch4-2组，分别转染si -NC 双链核苷酸、si -Notch4-1双链核苷酸、si -Notch4-2双链核苷酸，检测各组细胞株中Notch4 mRNA ，使用RTCA 法观察各组细胞株的增殖能力（CI 值），通过Transwell 实验观察各组细胞株的侵袭和迁移能力。

结果　与癌旁组织相比，口咽鳞癌组织中Notch4 mRNA 相对表达量高（P <0.01）；在对照细胞株HaCaT 和2BS 中Notch4 mRNA 相对表达量差异无统计学意义；与对照细胞株HaCaT 相比，口咽鳞癌细胞株Detroit 562、WSU -HN -30中Notch4 mRNA 相对表达量高（P 均<0.001）。

在口咽鳞癌细胞株Detroit 562、WSU -HN -30中，与si -NC 组相比，si -Notch4-1和si -Notch4-2组的细胞株增殖、迁移和侵袭能力降低（P 均<0.01）。

结论　Notch4 mRNA 在口咽鳞癌组织和细胞株中均高表达，敲降Notch4 mRNA 表达的口咽鳞癌细胞株增殖、迁移和侵袭能力降低。

Alpha-1-Proteinase Inhibitors产品说明

Alpha-1-Proteinase Inhibitors:Aralast NP®; Glassia®; Prolastin®-C; Zemaira®(Intravenous)Document Number: IC-0052 Last Review Date: 04/06/2021Date of Origin: 01/01/2012Dates Reviewed: 12/2011, 02/2013, 08/2013, 06/2014, 06/2015, 01/2016, 10/2016, 03/2017, 09/2017, 12/2017, 03/2018, 06/2018, 04/2019, 04/2020, 04/2021I.Length of AuthorizationCoverage will be provided for 12 months and may be renewed.II.Dosing LimitsA.Quantity Limit (max daily dose) [NDC Unit]:∙Aralast NP 1 g/50 mL: 7 vials per week∙Aralast NP 0.5 g/25 mL: 1 vial per week∙Glassia 1 g/50 mL: 7 vials per week∙Prolastin-C 1 g/20 mL: 7 vials per week∙Prolastin-C Liquid 1g/20 mL: 7 vials per week∙Zemaira 1 g/20 mL: 3 vials per week∙Zemaira 4 g/76 mL: 1 vial per week∙Zemaira 5 g/95 mL: 1 vial per weekB.Max Units (per dose and over time) [HCPCS Unit]:∙700 billable units every 7 daysIII.Initial Approval Criteria1-6,8,9,12•Patient is at least 18 years of age; ANDUniversal Criteria•Patient is not a tobacco smoker; AND•Patient is receiving optimal medical therapy (e.g., comprehensive case management, pulmonary rehabilitation, vaccinations, smoking cessation, self-management skills, etc.);AND•Patient does not have immunoglobulin-A (IgA) deficiency with antibodies against IgA;ANDEmphysema due to alpha-1-antitrypsin (AAT) deficiency †, Ф(applies only to Prolastin-C)•Patient has an FEV1 in the range of 30-65% of predicted; AND•Patient has alpha-1-antitrypsin (AAT) deficiency with PiZZ, PiZ (null), or Pi (null, null) phenotypes; AND•Patient has AAT-deficiency and clinical evidence of panacinar/panlobular emphysema;AND•Patient has low serum concentration of AAT ≤ 57 mg/dL or ≤ 11 µM/L as measured by nephelometry† FDA Approved Indication(s); Ф Orphan DrugIV.Renewal Criteria1-6,8,9Authorizations can be renewed based on the following criteria:•Patient continues to meet universal and other indication-specific relevant criteria such as concomitant therapy requirements (not including prerequisite therapy), performance status, etc.identified in section III; AND•Disease response with treatment as defined by elevation of AAT levels above baseline, substantial reduction in rate of deterioration of lung function as measured by percentpredicted FEV1, or improvement in CT scan lung density; AND•Absence of unacceptable toxicity from the drug. Examples of unacceptable toxicity include severe hypersensitivity reactions, etc.V.Dosage/Administration1-5VI.Billing Code/Availability InformationHCPCS Code & NDC:VII.References1.Glassia [package insert]. Lexington, MA; Baxalta US Inc.; June 2017. Accessed March2021.2.Zemaira [package insert]. Kankakee, IL; CSL Behring LLC; April 2019. Accessed March2021.3.Aralast NP [package insert]. Lexington, MA; Baxalta US Inc.; December 2018. AccessedMarch 2021.4.Prolastin-C Liquid [package insert]. Research Triangle Park, NC; Grifols Therapeutics,Inc.; August 2018. Accessed March 2021.5.Prolastin-C [package insert]. Research Triangle Park, NC; Grifols Therapeutics, Inc.; June2018. Accessed March 2021.6.American Thoracic Society/European Respiratory Society Statement: Standards for theDiagnosis and Management of Individuals with Alpha-1 Antitrypsin Deficiency. AmericanThoracic Society; European Respiratory Society. Am J Respir Crit Care Med. 2003 Oct1;168(7):818-900.7.Global Initiative for Chronic Obstructive Lung Disease (GOLD). Global strategy for thediagnosis, management, and prevention of chronic obstructive pulmonary disease. GlobalInitiative for Chronic Obstructive Lung Disease (GOLD); 2019.8.Sandhaus RA, Turino G, Brantly ML, et al. The diagnosis and management of alpha-1antitrypsin deficiency in the adult. Chronic Obstr Pulm Dis (Miami). 2016; 3(3):668-682.9.Marciniuk DD, Hernandez P, Balter M, et al. Alpha-1 antitrypsin deficiency targetedtesting and augmentation therapy: a Canadian Thoracic Society clinical practice guideline.Can Respir J. 2012;19(2):109-16.10.Stocks JM, Brantly M, Pollock D, et al. Multi-center study: the biochemical efficacy, safetyand tolerability of a new α1-proteinase inhibitor, Zemaira. COPD. 2006;3:17–23.11.Global Initiative for Chronic Obstructive Lung Disease (GOLD). Global strategy for thediagnosis, management, and prevention of chronic obstructive pulmonary disease. GlobalInitiative for Chronic Obstructive Lung Disease (GOLD); 2020.12.Miravitlles M, Dirksen A, Ferrarotti I, et al. European Respiratory Society statement:diagnosis and treatment of pulmonary disease in α1-antitrypsin deficiency. Eur Respir J2017; 50.Appendix 1 – Covered Diagnosis CodesAppendix 2 – Centers for Medicare and Medicaid Services (CMS)Medicare coverage for outpatient (Part B) drugs is outlined in the Medicare Benefit Policy Manual (Pub. 100-2), Chapter 15, §50 Drugs and Biologicals. In addition, National Coverage Determination (NCD), Local Coverage Determinations (LCDs), and Local Coverage Articles (LCAs) may exist and compliance with these policies is required where applicable. They can be found at: /medicare-coverage-database/search/advanced-search.aspx. Additional indications may be covered at the discretion of the health plan.Medicare Part B Covered Diagnosis Codes (applicable to existing NCD/LCD/LCA): N/A。

microRNA与肿瘤

作者单位： 200433 上海，第二军医大学附属长海医院消化内科通讯作者：李兆申，电子信箱： zhsti@ 81890． net
·10·
croRNAs，miRNAs）的发现，给肿瘤的研究带来了新的启示。miRNAs 与肿瘤的发生发展密切相关，被认为是一组新的致癌基因或抑癌基因［1 ～ 6］。本文就 microRNA 在肿瘤中的作用研究做一综述。
（收稿： 2010 － 09 － 21）（修回： 2011 － 01 － 21）
microRNA 与肿瘤
王卫卫李兆申高军邹多武
近年来对肿瘤的生物学标志进行了大量而广泛的研究，包括基因标志、血清学标志和细胞学标志等，由于这些指标的特异性低和敏感性差等原因，利用这些免疫标志物进行诊断仍有很多缺陷，需要有对肿瘤诊断有更好临床意义的指标。近年来，一类在转录水平上调控基因表达的非蛋白编码小分子 RNA （ mi-
医学研究杂志 2011 年 3 月第 40 卷第 3 期
·特别关注·
它由 19 ～ 23 个核苷酸组成，在细胞核中 RNA 核酸酶 Drosha － DGCR 复合体对 microRNA 初转录 pri － microRNA 的基部进行切割，形成 5'末端具有磷酸基，3″ 末端具有二核苷酸的茎环中间体－前体 microRNA （ pre － microRNA）。然后与转运蛋白 Exportin － 5 识别结合运输到细胞质。在胞质中 RNA 核酸酶 Dicer 识别 pre － microRNA 双链的 5'末端及 3'末端，在距茎环处进行切割，其中成熟的 microRNA 来源于 pre － microRNA 的一条臂，经过两次切割而成熟 microRNA 通过 RNA 诱导的沉默复合体（ RNA － induced silencing complex，RISC）在细胞中发挥作用［8，9］。

Split-GFP双分子荧光互补技术在鸡MSTN基因RNAi检测中的应用

54卷收稿日期：2023-04-27基金项目：国家自然科学基金项目（32060738，31660653）；广西自然科学基金重点项目（2018GXNSFDA281026）；广西重点研发计划项目（桂科AB16380098）通讯作者：郑喜邦（1964-），https:///0009-0006-7317-9381，博士，教授，主要从事家禽基因编辑研究工作，E-mail ：xibang-*************.cn 第一作者：杨磊（1995-），https:///0009-0006-0598-5410，研究方向为家禽转基因，E-mail ：*****************Split-GFP 双分子荧光互补技术在鸡MSTN 基因RNAi 检测中的应用杨磊，朱正，王博永，梁谦学，吴文德，李恭贺，郑喜邦*（广西大学动物科学技术学院，广西南宁530004）摘要：【目的】以Split-GFP 双荧光互补技术检测鸡肌肉生长抑制素基因（MSTN ）RNA 干涉（RNAi ）效果，并与其他常用检测方法比较，以验证Split-GFP 双分子荧光互补技术在RNAi 效果评估中的有效性和可行性。

【方法】将合成的3个shRNA 慢病毒载体（shRNA-a 、shRNA-b 和shRNA-c ）分别转染稳定表达GFP11-MSTN 融合蛋白的HEK 293T GFP11-MSTN 细胞，经实时荧光定量PCR 筛选出最佳shRNA 慢病毒载体并包装为慢病毒，然后以慢病毒感染HEK 293T GFP11-MSTN 细胞，采用潮霉素B 筛选mCherry 阳性（mCherry +）细胞，再以实时荧光定量PCR 和Western blotting 检测RNAi 效果；得到的mCherry +细胞再转染pcDNA3.1（+）-GFP1-10质粒，通过荧光显微镜观察和流式细胞术评估RNAi 效果。

【结果】3个shRNA 慢病毒载体对MSTN 基因表达均有极显著的抑制作用（P <0.01，下同），其中又以Anti-MSTN shRNA-a 慢病毒载体的干涉效果最佳。

ONX-0914 (PR-957)_免疫蛋白酶体(immunoproteasome)抑制剂_960374-59-8_Apexbio

请测试所有化合物在室内的溶解度，实际溶解度和理论值可能略有不同。这是由实验系统的误差引起的，属于正常现象。
参考文献: [1] Muchamuel T, Basler M, Aujay M A, et al. A selective inhibitor of the immunoproteasome subunit LMP7 blocks cytokine production and attenuates progression of experimental arthritis. Nature medicine, 2009, 15(7): 781-787.
动物实验: 动物模型
剂量应用
胶原抗体诱导的关节炎（CAIA）BALB/c 小鼠模型；胶原诱导的关节炎（CIA）DBA1/J 小鼠模型
2、6 和 10 mg/kg；静脉注射
ONX-0914 以剂量依赖的方式阻断疾病的发展，在最高剂量下完
注意事项
全改善疾病的明显迹象。PR-957 以 2 mg/kg 的剂量给药的治疗反应表明，LMP7 的单独抑制即可阻止疾病的发展。在 T 细胞和 B 细胞依赖的 CIA 模型中，ONX-0914 也诱导快速的治疗反应。免疫蛋白酶体的抑制与循环中自身抗体及胶原寡聚基质蛋白（COMP）水平的下降相关，COMP 是软骨破坏的标志物。
ONX-0914,PR-957
(2S)-3-(4-methoxyphenyl)-N-[(2S)-1-(2-methyloxiran-2-yl)-1-oxo-3-p henylpropan-2-yl]-2-[[(2S)-2-[(2-morpholin-4-ylacetyl)amino]propan oyl]amino]propanamide
实验操作

S1PR1介导的IFNAR1降解可以调节浆细胞样树突状细胞α-干扰素的自动扩增／信号放大(外文翻译)

S1PR1-mediated IFNAR1 degradation modulates plasmacytoiddendritic cell interferon-α autoamplification由S1PR1介导的IFNAR1降解可以调节浆细胞样树突状细胞α-干扰素的自动扩增／信号放大摘要：Blunting immunopathology without abolishing host defense is the foundation for safe and effective modulation of infectious and autoimmune diseases.没有废除宿主防御机制的免疫病理钝化是安全、有效调节传染病和自身免疫性疾病的基础。

Sphingosine 1-phosphate receptor 1 (S1PR1) agonists are effective in treating infectious and multiple autoimmune pathologies; however, mechanisms underlying their clinical efficacy are yet to be fully elucidated.1-磷酸-鞘氨醇受体1（S1PR1）促效药对于治疗传染病和多种自身免疫性疾病是有效的，然而，其临床疗效的具体机制尚未被完全阐明。

Here, we uncover an unexpected mechanism of convergence between S1PR1 and interferon alpha receptor 1 (IFNAR1) signaling pathways.在本研究中，我们意外发现S1PR1与α-干扰素受体1（IFNAR1）信号通路之间的趋同／聚集机制。

Activation of S1PR1 signaling by pharmacological tools or endogenous ligand sphingosine-1 phosphate (S1P) inhibits type 1 IFN responses that exacerbate numerous pathogenic conditions.通过药理作用或内源性配体1-磷酸-鞘氨醇（S1P）发出信号激活S1PR1可以抑制1型干扰素应答，这将提供大量致病条件。

Ubiquitin-Proteasome Pathway

Ubiquitin-Proteasome Pathway
SIGMA-ALDRICH
Ubiquitin-Proteasome Pathway
Intracellular proteolytic systems recognize and destroy misfolded or damaged proteins, unassembled polypeptide chains, and short-lived regulatory proteins. There are several mechanisms for protein degradation within cells. Two systems that play important roles in proteolysis resulting from cell stress are the calpain proteases and the ubiquitin-proteasome pathway. The ubiquitin-proteasome pathway functions widely in intracellular protein turnover. It plays a central role in degradation of short-lived and regulatory proteins important in a variety of basic cellular processes, including regulation of the cell cycle, modulation of cell surface receptors and ion channels, and antigen processing and presentation. The pathway employs an enzymatic cascade by which multiple ubiquitin molecules are covalently attached to the protein substrate. The polyubiquitin modification marks the protein for destruction and directs it to the 26S proteasome complex for degradation. References Sommer, T., et al., Compartment - specific functions of the ubiquitin - proteasome pathway. Rev. Physiol. Biochem. Pharmacol., 142, 97-160 (2001). Doskeland, A.P., and Flatmark, T., Cowith polyubiquitin chains catalysed by rat liver enzymes. Biochim. Biophys. Acta., 1547, 379-386 (2001). Hartmann-Petersen, R., et al., Quaternary structure of the ATPase complex of human 26S proteasomes determined by chemical cross-linking. Arch. Biochem. Biophys., 386, 89-94 (2001). Brooks, P., et al., Subcellular localization of proteasomes and their regulatory complexes in mammalian cells. Biochem. J., 346, 155-161 (2000).

Prdx1与肿瘤

Prdx1与肿瘤高军;朱虹;高国兰【摘要】Peroxiredoxin 1(Prdx1)belongs to peroxide oxidoreductase protein(peroxiredoxin, Prdx)family,which is over-expressed in multiple cancers and it play an important role in antagonizing reactive oxygen species (ROS) therefore as works as anti-oxidations. Its expression is closely related with tumor proliferation, differentiation and metastasis as well as with sensibility to radiotherapy and chemotherapy. Its structure and biological function in tumors and regulation mechanism were reviewed in this paper. It can provide evidence to screen new therapeutic targets in the treatment of tumors.%Prdx1（peroxiredoxin 1）属于过氧化物氧化还原酶蛋白（peroxiredoxin，Prdx）家族成员，是一类在对抗活性氧簇（ROS）、抗氧化过程中发挥关键作用的蛋白质，在多种肿瘤组织中高表达，其表达与肿瘤细胞增殖、分化、转移、复发及放化疗的敏感性密切相关。

本文就Prdx1的结构、在肿瘤中的生物学功能及其信号调控机制等进行综述，为寻找新的肿瘤生物治疗靶点提供依据。

【期刊名称】《天津医药》【年(卷),期】2015(000)012【总页数】3页(P1464-1466)【关键词】Prdx1;过氧化物酶;肿瘤【作者】高军;朱虹;高国兰【作者单位】江西省人民医院妇科邮编330006;江西省人民医院妇科邮编330006;中国医科大学航空总医院妇科【正文语种】中文【中图分类】R730.2;R73-3众所周知，生命的进化经历了无氧至有氧的发展过程。

非编码RNA来源的小肽：“微不足道”却“功能强大”

第 62 卷第 3 期2023 年 5 月Vol.62 No.3May 2023中山大学学报（自然科学版）（中英文）ACTA SCIENTIARUM NATURALIUM UNIVERSITATIS SUNYATSENI非编码RNA来源的小肽：“微不足道”却“功能强大”*陈晓彤，赵文龙，孙林玉，王文涛，陈月琴中山大学生命科学学院，广东广州 510275摘要：非编码RNA（ncRNA， non-coding RNA）长久以来被认为不具有编码能力。

近年来随着研究技术和生物信息学工具的迅速发展，研究发现在基因组的非编码区域上存在大量小开放阅读框（sORFs，small/short open read‐ing frames），其翻译产物被称作小ORF编码肽（SEPs，sORF encoded peptides）或小肽（micropeptides）。

部分小肽被证实在细胞内稳定存在并独立于其来源RNA发挥重要作用。

本文系统总结了非编码RNA来源小肽的鉴定方法、可编码小肽的RNA类型以及其研究困难和瓶颈，并重点回顾了疾病和植物中发现的功能小肽，以期对小肽的筛选鉴定提供思考，对小肽作为药物研发或者农作物增产的关键靶点提供新的思路和方向。

关键词：非编码RNA；小肽；非经典翻译；鉴定方法；调控机制中图分类号：Q71 文献标志码：A 文章编号：2097 - 0137（2023）03 - 0001 - 13 Micropeptides derived from non-coding RNAs： Tiny but powerful CHEN Xiaotong， ZHAO Wenlong， SUN Linyu， WANG Wentao， CHEN Yueqin School of Life Sciences， Sun Yat-sen University， Guangzhou 510275，ChinaAbstract：It was long presumed that non-coding RNAs （ncRNAs） are lacking in protein-coding poten‐tial. However， recent advances in technology and tools have led to an important finding that a number of small open reading frames （sORFs） were found in different kind of ncRNAs， and their translated products have been termed sORF encoded peptides （SEPs） or micropeptides. Some micropeptides have been confirmed to exist stably in cells and play important roles independently of their source RNA. In this review，we summarize the identification methods of micropeptides derived from ncRNAs，the types of RNA that can encode micropeptides，and focus on the functional micropeptides found in diseases and plants. The purpose of the review is to provide a thought on the screening and identifica‐tion of micropeptides， and provide new ideas for micropeptides as potentials for drug development or crop yield improvement.Key words： non-coding RNA; micropeptide; non-canonical translation; identification methods; regula‐tion mechanism随着人类基因组计划的完成以及ENCODE计划的开展，科学家发现，约75%的基因组可以产生转录本（Derrien et al.，2012；Djebali et al.，2012）。

lipofectamineRNAiMAX英文说明书

Lipofectamine™ RNAiMAXCat. No. 13778-075 Size: 0.75 mlCat. No. 13778-150 Size: 1.5 mlStore at +4°C (do not freeze) DescriptionLipofectamine™ RNAiMAX is a proprietary formulation specifically developed for the transfection of siRNA and Stealth™ RNAi duplexes into eukaryotic cells. Lipofectamine™ RNAiMAX provides the following advantages:• High transfection efficiencies in many cell types to minimize background expression from untransfected cells and maximize knockdown.• Minimal cytotoxicity to reduce non-specific effects and cellular stress.• Generally requires low concentrations of RNAi duplexes to obtain high knockdown levels, further minimizing non-specific effects.• A broad peak of optimal transfection activity with minimal cytotoxicity, allowing achievement of high knockdown levels despite differences in cell density, minor pipetting inaccuracies, and other variations.Important Guidelines for Transfection• Reverse transfection (page 2) and forward transfection (page 3) protocols can be used for most cell lines tested. Cell-type specific transfection protocols are available at /RNAi or through Technical Service.• We recommend Opti-MEM® I Reduced Serum Medium (Cat. No. 31985-062) to dilute RNAi duplexes and Lipofectamine™ RNAiMAX before complexing.• Do not add antibiotics to media during transfection as this causes cell death.• Test serum-free media for compatibility with Lipofectamine™ RNAiMAX. • To assess transfection efficiency, we recommend using a KIF11 Stealth™Select RNAi, as described in Assessing Transfection Efficiency (page 2). • Use 10 nM RNAi duplex and indicated procedure as a starting point;optimize transfections as described in Optimizing Transfections (page 3). Quality ControlLipofectamine™ RNAiMAX is tested for absence of microbial contamination with blood agar plates, Sabaraud dextrose agar plates, and fluid thioglycolate medium, for absence of RNAse activity, and functionally by transfection of Stealth™ RNAi and appropriate controls into a reporter cell line.Part No.: 13778.PPS Rev. Date: 11 Jan 2006For research use only. Not intended for any animal or human therapeutic or diagnostic use.For technical support, contact tech_service@.Page 2 Reverse TransfectionUse this procedure to reverse transfect Stealth™ RNAi or siRNA into mammalian cells in a 24-well format (for other formats, see Scaling Up or Down Transfections, page 4). In reverse transfections, the complexes are prepared inside the wells, after which cells and medium are added. Reverse transfections are faster to perform than forward transfections, and are the method of choice for high-throughput transfection. Optimize transfections as described in Optimizing Transfections (page 3), especially if transfecting a mammalian cell line for the first time. All amounts and volumes are given on a per well basis.1. For each well to be transfected, prepare RNAi duplex-Lipofectamine™RNAiMAX complexes as follows.a. Dilute 6 pmol RNAi duplex in 100 µl Opti-MEM® I Medium without serumin the well of the tissue culture plate. Mix gently.b. Mix Lipofectamine™ RNAiMAX gently before use, then add 1 µlLipofectamine™ RNAiMAX to each well containing the diluted RNAimolecules. Mix gently and incubate for 10-20 minutes at roomtemperature.2. Dilute cells in complete growth medium without antibiotics so that 500 µlcontains the appropriate number of cells to give 30-50% confluence 24 hours after plating. Use 20,000-50,000 cells/well for suspension cells.3. To each well with RNAi duplex - Lipofectamine™ RNAiMAX complexes, add500 µl of the diluted cells. This gives a final volume of 600 µl and a final RNA concentration of 10 nM. Mix gently by rocking the plate back and forth.4. Incubate the cells 24-72 hours at 37°C in a CO2 incubator until you are readyto assay for gene knockdown.Assessing Transfection EfficiencyTo qualitatively assess transfection efficiency, we recommend using a KIF11 Stealth™ Select RNAi (available through /rnaiexpress; for human cells, oligo HSS105842 is a good choice). Adherent cells in whichKIF11/Eg5 is knocked down exhibit a “rounded-up” phenotype after 24 hours due to a mitotic arrest (Weil, D. et al., Biotechniques (2002), 33: 1244-1248); slow growing cells may take up to 72 hours to display the rounded phenotype. Alternatively, growth inhibition can be assayed after 48-72 hours.Note: The BLOCK-iT™ Fluorescent Oligo (Cat. No. 2013) is optimized for use with Lipofectamine™ 2000, and is not recommended for Lipofectamine™ RNAiMAX.Page 3 Forward TransfectionUse this procedure to forward transfect Stealth™ RNAi or siRNA into mammalian cells in a 24-well format (for other formats, see Scaling Up or Down Transfections, page 4). In forward transfections, cells are plated in the wells, and the transfection mix is generally prepared and added the next day. Optimize transfections as described in Optimizing Transfections (page 3), especially if transfecting a mammalian cell line for the first time. All amounts and volumes are given on a per well basis.Note: For some cell lines (e.g. MCF-7 or HepG2), we recommend reverse transfections.1. One day before transfection, plate cells in 500 µl of growth medium withoutantibiotics such that they will be 30-50% confluent at the time of transfection.2. For each well to be transfected, prepare RNAi duplex-Lipofectamine™RNAiMAX complexes as follows:a. Dilute 6 pmol RNAi duplex in 50 µl Opti-MEM®I Reduced Serum Mediumwithout serum. Mix gently.b. Mix Lipofectamine™ RNAiMAX gently before use, then dilute 1 µl in 50 µlOpti-MEM® I Reduced Serum Medium. Mix gently.c. Combine the diluted RNAi duplex with the diluted Lipofectamine™RNAiMAX. Mix gently and incubate for 10-20 minutes at roomtemperature.3. Add the RNAi duplex-Lipofectamine™ RNAiMAX complexes to each wellcontaining cells. This gives a final volume of 600 µl and a final RNAconcentration of 10 nM. Mix gently by rocking the plate back and forth.4. Incubate the cells 24-48 hours at 37°C in a CO2 incubator until you areready to assay for gene knockdown. Medium may be changed after 4-6hours.Optimizing TransfectionsTo obtain the highest transfection efficiency and low non-specific effects, optimize transfection conditions by varying RNAi duplex and Lipofectamine™RNAiMAX concentrations. Test 0.6-30 pmol RNAi duplex (final concentration 1-50 nM) and 0.5-1.5 µl Lipofectamine™ RNAiMAX for 24-well format. For extended time course experiments (> 72 hours), consider a cell density that is 10-20% confluent 24 hours after plating.Note: The concentration of RNAi duplex required will vary depending on the efficacy of the duplex.Page 4 Scaling Up or Down TransfectionsTo transfect cells in different tissue culture formats, vary the amounts of Lipofectamine™ RNAiMAX, RNAi duplex, cells, and medium used in proportion to the relative surface area, as shown in the table.Culture vessel Rel.surf.area1Vol. ofplatingmediumDilutionmediumreversetransfectionDilutionmediumforwardtransfectionRNAi(pmol)RNAi(nM)Lipofect-amine™RNAiMAX296-well 0.2 100 µl 20 µl 2 x 10 µl 0.12-6 1-50 0.1-0.3 µl48-well 0.4 200 µl 40 µl 2 x 20 µl 0.24-12 1-50 0.2-0.6 µl24-well 1 500 µl 100 µl 2 x 50 µl 0.6-30 1-50 0.5-1.5 µl6-well 5 2.5 ml 500 µl 2 x 250 µl 3-150 1-50 2.5-7.5 µl60 mm 10 5 ml 1 ml 2 x 500 µl 6-300 1-50 5-15 µl100 mm 30 10 ml 2 ml 2 x 1 ml 12-600 1-50 15-35 µl1 Surface areas may vary depending on the manufacturer.2If the volume of Lipofectamine™ RNAiMAX is too small to dispense accurately, and you cannot pool dilutions, predilute Lipofectamine™ RNAiMAX 10-fold in Opti-MEM®I Reduced Serum Medium, and dispense a 10-fold higher amount (should be at least1.0 µl per well). Discard any unused diluted Lipofectamine™ RNAiMAX. Cotransfecting DNA and RNA using Lipofectamine™ RNAiMAX For cotransfections of plasmid DNA and Stealth™ RNAi or siRNA into mammalian cells, we recommend using Lipofectamine™ 2000 (Catalog no. 11668-027), which is superior for plasmid transfections. If you want to use Lipofectamine™ RNAiMAX for your cotransfections, perform a reverse transfection as described on page 2 with the following modifications:1a: Add 20 ng (for 24-well format) of plasmid DNA to the diluted RNAi duplex. 2: Add cells such that they will be 80-100% confluent 24 hours after plating.Purchaser NotificationThis product is covered by one or more Limited Use Label Licenses (see the Invitrogen catalog or our web-site, ). By the use of this product you accept the terms and conditions of all applicable Limited Use Label Licenses.Limited Use Label License No. 5Limited Use Label License No. 27©2006 Invitrogen Corporation. All rights reserved.。

构建诱导型CRISPRCas9系统用于小鼠免疫细胞基因功能研究

Vol.41No.3Mar.2021上海交通大学学报（医学版）JOURNAL OF SHANGHAI JIAO TONG UNIVERSITY (MEDICAL SCIENCE)构建诱导型CRISPR/Cas9系统用于小鼠免疫细胞基因功能研究赵艳娜1*，邱荣2*，沈南1，唐元家11.上海交通大学医学院附属仁济医院风湿病科，上海市风湿病学研究所，上海200127；2.中国科学院上海营养与健康研究所，上海200031［摘要］目的·结合Dox 诱导型单链导向RNA （single guide RNA ，sgRNA ）表达载体和Cas9转基因小鼠，构建诱导型CRISPR/Cas9系统用于小鼠免疫细胞基因功能研究。

方法·根据四环素诱导表达系统原理，基因合成U6-TetO-sgRNA 和EF1α-T2A-Puro-BFP-T2A-TetR 片段。

通过同源重组将2个片段组装进反转录病毒载体骨架，获得Dox 诱导型sgRNA 反转录病毒载体。

为了验证系统有效性，分离Cas9转基因小鼠骨髓细胞并诱导其向巨噬细胞方向分化。

设计对照（non-targeting control ，NC ）组和实验组（靶向F4/80）的sgRNA ，利用反转录病毒感染细胞，分化条件设置添加Dox 组（Dox +）和不添加Dox 组（Dox -）。

通过流式细胞术和T7核酸内切酶Ⅰ（T7endonuclease Ⅰ，T7E Ⅰ）实验检测基因敲除效果。

结果·①成功构建Dox 诱导型sgRNA 反转录病毒表达载体和Cas9转基因小鼠。

②流式结果显示，在NC Dox -组、NC Dox +组和F4/80Dox -组中，几乎无F4/80阴性细胞群体；而在F4/80Dox +组中，F4/80阴性细胞群体高达50%。

③T 7E Ⅰ结果显示，在F4/80Dox -组中，DNA 条带完整，而在F4/80Dox +组中发生基因突变，DNA 条带被切开。

莲草直胸跳甲短神经肽F受体基因sNPFR的克隆及表达分析

山西农业科学 2024，52（1）：137-144Journal of Shanxi Agricultural Sciences 莲草直胸跳甲短神经肽F受体基因sNPFR的克隆及表达分析王康，赵雪莹，霍楠，胡军，王苑馨，杨军，贾栋（山西农业大学植物保护学院，山西太谷 030801）摘要：为明确莲草直胸跳甲（Agasicles hygrophila）短神经肽F受体AhsNPFR功能及其表达特点，为探索莲草直胸跳甲生防作用奠定理论基础，利用PCR技术克隆鉴定莲草直胸跳甲AhsNPFR基因并进行生物信息学分析，通过实时荧光定量PCR技术分析其在莲草直胸跳甲不同发育时期和组织中的时空表达谱。

结果表明，克隆获得基因AhsNPFR全长1 669 bp，开放阅读框1 257 bp，编码418个氨基酸；预测其蛋白质分子质量为48.11 ku，理论等电点为8.21，AhsNPFR具有7个典型保守跨膜结构域，属于GPCRs家族，系统进化树分析表明，其与玉米根萤叶甲Diabrotica virgifera virgifera sNPFR亲缘关系最近。

AhsNPFR基因在不同发育阶段均有表达，在1龄幼虫中的表达量最高，是卵中表达量的9.06倍；在卵中表达量最低；雌成虫的表达量显著高于雄成虫。

AhsN⁃PFR基因在不同组织中均有表达，在3龄幼虫后肠中显著高表达，是脂肪体表达量的12.21倍；在雌雄成虫后肠显著高表达，并在所有组织中均没有雌雄表达差异。

关键词：莲草直胸跳甲；短神经肽F受体基因sNPFR；基因克隆；发育时期表达；组织表达中图分类号：S476.2　文献标识码：A 文章编号：1002‒2481（2024）01‒0137‒08Cloning and Expression Profiling of Short Neuropeptide F ReceptorGene sNPFR in Agasicles hygrophilaWANG Kang，ZHAO Xueying，HUO Nan，HU Jun，WANG Yuanxin，YANG Jun，JIA Dong （College of Plant Protection，Shanxi Agricultural University，Taigu 030801，China）Abstract：To explore the function and expression characteristics of the short neuropeptide receptor AhsNPFR in Agasicles hygrophila, and to lay a theoretical foundation for exploring the biological control role of Agasicles hygrophila, in this study, the AhsNPFR gene was cloned using PCR technology and analyzed with bioinformatics software. The spatiotemporal expression profiles of AhsNPFR across different developmental stages and tissues were investigated by quantitative real-time PCR(qPCR). The results showed that the complete sequence of 1 669 bp was obtained, which was found to contain a 1 257 bp open reading frame(ORF), encoding 418 amino acids. The predicted molecular weight was 48.11 ku and the theoretical isoelectric point(pI) was 8.21. AhsNPFR had 7 typical conserved transmembrane domains and belonged to the GPCRs family. Phylogenetic tree analysis showed that AhsNPFR was closely related to Diabrotica virgifera virgifera sNPFR. AhsNPFR gene was expressed throughout the developmental stages tested with the highest expression levels at the first instar larva which was 9.06 times higher than those eggs, the stage with the lowest expression level. The expression level in female adult was significantly higher than that in male adult. The AhsNPFR gene was expressed in different tissues and was significantly highly expressed in the hindgut of the 3rd instar larvae, which was 12.21 times higher than those fat body. The AhsNPFR gene was significantly highly expressed in the hindgut of male and female adults, with no difference expression between the sexes across all tissues.Key words：Agasicles hygrophila; short neuropeptide F receptor gene sNPFR; gene cloning; developmental stage expres⁃sion; tissue expression神经肽是一类重要的信号分子，在昆虫的生长发育、繁殖和行为等多种生物学过程中发挥重要调节作用[1]。

n端降解子通路

n端降解子通路
"N端降解子通路"通常指的是蛋白质的降解过程中涉及的一种途径。

蛋白质降解是指蛋白质分子被分解成较小的片段或氨基酸的过程，这是细胞中维持蛋白质平衡和清除受损或不需要的蛋白质的重要机制之一。

N端降解子通路主要涉及以下两个主要系统：
* 封闭N端降解子系统（Ubiquitin-Proteasome System，UPS）：这是一种广泛存在于真核生物细胞中的蛋白质降解系统。

在这个系统中，目标蛋白质的N端被标记上小的多肽标签，即泛素（ubiquitin）。

泛素附着到蛋白质上，然后这个被泛素标记的蛋白质被送入蛋白酶体（proteasome）进行降解。

这个过程对于调控细胞周期、细胞信号传导和蛋白质质量控制非常重要。

 
* 酸性N端降解子系统（Autophagy）：这是一种通过溶酶体降解蛋白质的过程。

在细胞发现有损或不需要的细胞器、蛋白质聚集等时，细胞通过自噬的方式将这些结构包裹成囊泡，然后将其运送到溶酶体进行降解。

这个过程对于清除细胞内的损伤物质、回收有用物质以及维持细胞稳态也至关重要。

 
这两个降解系统协同工作，维持了细胞内蛋白质的平衡，对于细胞的正常功能和生存至关重要。