Current Protocols in Molecular Biology最新文献_第10页

Avian Retrovirus-Mediated Tumor-Specific Gene Knockout 禽逆转录病毒介导的肿瘤特异性基因敲除

Current Protocols in Molecular Biology Pub Date : 2018-01-16 DOI: 10.1002/cpmb.54

Wei Wang, Bingning Dong, Feng Yang

引用次数: 2

Modulating Gene Expression in Epstein-Barr Virus (EBV)-Positive B Cell Lines with CRISPRa and CRISPRi 用CRISPRa和CRISPRi调控eb病毒阳性B细胞株的基因表达

Current Protocols in Molecular Biology Pub Date : 2018-01-16 DOI: 10.1002/cpmb.50

Liang Wei Wang, Stephen J. Trudeau, Chong Wang, Catherine Gerdt, Sizun Jiang, Bo Zhao, Benjamin E. Gewurz

{"title":"Modulating Gene Expression in Epstein-Barr Virus (EBV)-Positive B Cell Lines with CRISPRa and CRISPRi","authors":"Liang Wei Wang, Stephen J. Trudeau, Chong Wang, Catherine Gerdt, Sizun Jiang, Bo Zhao, Benjamin E. Gewurz","doi":"10.1002/cpmb.50","DOIUrl":"10.1002/cpmb.50","url":null,"abstract":"Epstein-Barr virus (EBV) transforms small resting primary B cells into large lymphoblastoid cells which are able to grow and survive in vitro indefinitely. These cells represent a model for oncogenesis. In this unit, variants of conventional clustered regularly interspaced short palindromic repeats (CRISPR), namely the CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) methods, are discussed in the context of gene regulation at genomic DNA promoter and enhancer elements. Lymphoblastoid B cell lines (LCLs) stably expressing nuclease-deficient Cas9 (dCas9)-VP64 (Cas9 associated with CRISPRa) or dCas9-KRAB (Cas9 associated with CRISPRi) are transduced with lentivirus that encodes a single guide RNA (sgRNA) that targets a specific gene locus. The ribonucleoprotein complex formed by the dCas9 molecule and its cognate sgRNA enables sequence-specific binding at a promoter or enhancer of interest to affect the expression of genes regulated by the targeted promoter or enhancer. © 2018 by John Wiley & Sons, Inc.","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"121 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-01-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmb.50","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35739637","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 6

Making Use of Cancer Genomic Databases 利用癌症基因组数据库

Current Protocols in Molecular Biology Pub Date : 2018-01-16 DOI: 10.1002/cpmb.49

Chad J. Creighton

引用次数: 14

Single-Assay Profiling of Nucleosome Occupancy and Chromatin Accessibility. 核小体占用和染色质可及性的单分析分析。

Current Protocols in Molecular Biology Pub Date : 2017-10-02 DOI: 10.1002/cpmb.45

April Cook, Jakub Mieczkowski, Michael Y Tolstorukov

引用次数: 7

CASAAV: A CRISPR-Based Platform for Rapid Dissection of Gene Function In Vivo. CASAAV：基于 CRISPR 的体内基因功能快速剖析平台。

Current Protocols in Molecular Biology Pub Date : 2017-10-02 DOI: 10.1002/cpmb.46

Nathan J VanDusen, Yuxuan Guo, Weiliang Gu, William T Pu

引用次数: 0

Production of Purified CasRNPs for Efficacious Genome Editing. 生产纯化的 CasRNPs 以实现有效的基因组编辑。

Current Protocols in Molecular Biology Pub Date : 2017-10-02 DOI: 10.1002/cpmb.43

Emily Lingeman, Chris Jeans, Jacob E Corn

引用次数: 30

Transposon Insertion Site Sequencing (TIS-Seq): An Efficient and High-Throughput Method for Determining Transposon Insertion Site(s) and Their Relative Abundances in a PiggyBac Transposon Mutant Pool by Next-Generation Sequencing. 转座子插入位点测序(TIS-Seq):利用新一代测序技术测定PiggyBac转座子突变体库中转座子插入位点及其相对丰度的高效、高通量方法。

Current Protocols in Molecular Biology Pub Date : 2017-10-02 DOI: 10.1002/cpmb.47

Yaligara Veeranagouda, Michel Didier

{"title":"Transposon Insertion Site Sequencing (TIS-Seq): An Efficient and High-Throughput Method for Determining Transposon Insertion Site(s) and Their Relative Abundances in a PiggyBac Transposon Mutant Pool by Next-Generation Sequencing.","authors":"Yaligara Veeranagouda, Michel Didier","doi":"10.1002/cpmb.47","DOIUrl":"https://doi.org/10.1002/cpmb.47","url":null,"abstract":"The PiggyBac (PB) transposon has emerged as a novel mutagenesis tool for understanding gene function and for phenotypic screening in eukaryotes. Successful screening of PB transposon mutants relies on efficient identification of transposon insertion site(s) (TIS) in mutant cells. However, currently available methods suffer from time-consuming steps. Here, we present the method for transposon insertion site sequencing (TIS-Seq) for high-throughput identification of TIS in transposon mutants. TIS-Seq provides qualitative and quantitative information on mutants present in a given PB transposon mutant library. TIS-Seq also facilitates identification of TIS in up to 96 individual/hand-picked mutants in a single MiniSeq/MiSeq run. TIS-Seq is a versatile method that can be easily modified to identify TIS from any kind of transposon mutant, as long as one end of the DNA sequence is known. Therefore, TIS-Seq is a promising method for transposon mutant screening. © 2017 by John Wiley & Sons, Inc.","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"120 ","pages":"21.35.1-21.35.11"},"PeriodicalIF":0.0,"publicationDate":"2017-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmb.47","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35460054","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 7

Mapping Transposon Insertions in Bacterial Genomes by Arbitrarily Primed PCR 利用任意引物PCR定位细菌基因组中的转座子插入

Current Protocols in Molecular Biology Pub Date : 2017-04-03 DOI: 10.1002/cpmb.38

José T. Saavedra, Julia A. Schwartzman, Michael S. Gilmore

{"title":"Mapping Transposon Insertions in Bacterial Genomes by Arbitrarily Primed PCR","authors":"José T. Saavedra, Julia A. Schwartzman, Michael S. Gilmore","doi":"10.1002/cpmb.38","DOIUrl":"10.1002/cpmb.38","url":null,"abstract":"Transposons can be used to easily generate and label the location of mutations throughout bacterial and other genomes. Transposon insertion mutants may be screened for a phenotype as individual isolates, or by selection applied to a pool of thousands of mutants. Identifying the location of a transposon insertion is critical for connecting phenotype to the genetic lesion. In this unit, we present an easy and detailed approach for mapping transposon insertion sites using arbitrarily-primed PCR (AP-PCR). Two rounds of PCR are used to (1) amplify DNA spanning the transposon insertion junction, and (2) increase the specific yield of transposon insertion junction fragments for sequence analysis. The resulting sequence is mapped to a bacterial genome to identify the site of transposon insertion. In this protocol, AP-PCR as it is routinely used to map sites of transposon insertion within Staphylococcus aureus, is used to illustrate the principle. Guidelines are provided for adapting this protocol for mapping insertions in other bacterial genomes. Mapping transposon insertions using this method is typically achieved in 2 to 3 days if starting from a culture of the transposon insertion mutant. © 2017 by John Wiley & Sons, Inc.","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"118 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmb.38","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"34879180","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 16

Direct Isolation of Seamless Mutant Bacterial Artificial Chromosomes 无缝突变细菌人工染色体的直接分离

Current Protocols in Molecular Biology Pub Date : 2017-04-03 DOI: 10.1002/cpmb.34

George T. Lyozin, Yasuhiro Kosaka, Gourab Bhattacharje, H. Joseph Yost, Luca Brunelli

{"title":"Direct Isolation of Seamless Mutant Bacterial Artificial Chromosomes","authors":"George T. Lyozin, Yasuhiro Kosaka, Gourab Bhattacharje, H. Joseph Yost, Luca Brunelli","doi":"10.1002/cpmb.34","DOIUrl":"10.1002/cpmb.34","url":null,"abstract":"Seamless (i.e., without unwanted DNA sequences) mutant bacterial artificial chromosomes (BACs) generated via recombination-mediated genetic engineering (recombineering) are better suited to study gene function compared to complementary DNA (cDNA) because they contain only the specific mutation and provide all the regulatory sequences required for in vivo gene expression. However, precisely mutated BACs are typically rare (∼1:1,000 to 1:100,000), making their isolation quite challenging. Although these BACs have been classically isolated by linking the mutation to additional genes, i.e., selectable markers, this approach is prone to false positives and is labor-intensive because it requires the subsequent removal of the selectable marker. We created Founder Principle–driven Enrichment (FPE), a method based on the population genetics “founder principle,” to directly isolate rare mutant BACs, without any selectable marker, from liquid cultures via the polymerase chain reaction (PCR). Here, we provide a detailed description of FPE, including protocols for BAC recombineering and PCR screening. © 2017 by John Wiley & Sons, Inc.","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"118 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmb.34","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"34879252","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 4

Transcriptome-wide Identification of RNA-binding Protein Binding Sites Using Photoactivatable-Ribonucleoside-Enhanced Crosslinking Immunoprecipitation (PAR-CLIP) 利用光活化核糖核苷增强交联免疫沉淀(PAR-CLIP)在转录组范围内鉴定rna结合蛋白结合位点

Current Protocols in Molecular Biology Pub Date : 2017-04-03 DOI: 10.1002/cpmb.35

Henrike Maatz, Marcin Kolinski, Norbert Hubner, Markus Landthaler

{"title":"Transcriptome-wide Identification of RNA-binding Protein Binding Sites Using Photoactivatable-Ribonucleoside-Enhanced Crosslinking Immunoprecipitation (PAR-CLIP)","authors":"Henrike Maatz, Marcin Kolinski, Norbert Hubner, Markus Landthaler","doi":"10.1002/cpmb.35","DOIUrl":"10.1002/cpmb.35","url":null,"abstract":"RNA‐binding proteins (RBPs) mediate important co‐ and post‐transcriptional gene regulation by binding pre‐mRNA in a sequence‐ and/or structure‐specific manner. For a comprehensive understanding of RBP function, transcriptome‐wide mapping of the RNA‐binding sites is essential, and CLIP‐seq methods have been developed to elucidate protein/RNA interactions at high resolution. CLIP‐seq combines protein/RNA UV‐crosslinking with immunoprecipitation (CLIP) followed by high‐throughput sequencing of crosslinked RNA fragments. To overcome the limitations of low RNA‐protein crosslinking efficiency in standard CLIP‐seq, photoactivatable‐ribonucleoside‐enhanced CLIP (PAR‐CLIP) has been developed. Here, living cells or whole organisms are fed photo‐activatable nucleoside analogs that are incorporated into nascent RNA transcripts before UV treatment. This allows greater crosslinking efficiency at comparable radiation doses for enhanced RNA recovery and separation of crosslinked target RNA fragments from background RNA degradation products. Moreover, it facilitates the generation of specific UV‐induced mutations that mark the crosslinking nucleotide and allow transcriptome‐wide identification of RBP binding sites at single‐nucleotide resolution. © by 2017 John Wiley & Sons, Inc.","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"118 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmb.35","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"34879251","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 10