Bio-protocol最新文献

{"title":"Accurate Measurement of Cell Number-Normalized Differential Gene Expression in Cells Treated With Retinoic Acid.","authors":"Nina Weichert-Leahey, Mark W Zimmerman, Alla Berezovskaya, A Thomas Look, Brian J Abraham","doi":"10.21769/BioProtoc.5106","DOIUrl":"https://doi.org/10.21769/BioProtoc.5106","url":null,"abstract":"<p><p>Genome-wide gene expression analysis is a commonly used method to quantitatively examine the transcriptional signature of any tissue or cell state. Standard bulk cell RNA sequencing (RNA-seq) quantifies RNAs in the cells of the tissue type of interest through massive parallel sequencing of cDNA synthesized from the cellular RNA. The subsequent analysis of global RNA expression and normalization of RNA expression levels between two or more samples generally assumes that cells from all samples produce equivalent amounts of RNA per cell. This assumption may be invalid in cells where <i>MYC</i> or <i>MYCN</i> expression levels are markedly different and thus, overall mRNA expression per cell is altered. Here, we describe an approach for RNA-seq analysis of <i>MYCN</i>-amplified neuroblastoma cells during treatment with retinoic acid, which causes dramatic downregulation of <i>MYCN</i> expression and induces growth arrest and differentiation of the cells. Our procedure employs spiked-in RNA standards added in ratio to the number of cells in each sample prior to RNA extraction. In the analysis of differential gene expression, the expression level of each gene is standardized to the spiked-in RNA standard to accurately assess gene expression levels per cell in conditions of high and low MYCN expression. Our protocol thus provides a step-by-step experimental approach for normalizing RNA-seq expression data on a per-cell-number basis, allowing accurate assessment of differential gene expression in cells expressing markedly different levels of MYC or MYCN. Key features • High levels of <i>MYC</i> and <i>MYCN</i> expression in cancer cells cause substantial increases in the levels of overall mRNA expression per cell. • RNA-seq using control RNAs spiked-in on a per-cell basis more accurately reflects global expression changes, when comparing cell populations with substantially different <i>MYCN</i> expression levels. • In <i>MYCN</i>-amplified neuroblastoma, retinoic acid dramatically decreases <i>MYCN</i> expression levels, resulting in large changes in overall RNA expression levels per cell.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"14 21","pages":"e5106"},"PeriodicalIF":1.0,"publicationDate":"2024-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11543784/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142634117","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Generation of Zebrafish Maternal Mutants via Oocyte-Specific Knockout System.","authors":"Chong Zhang, Wenlu Wei, Tong Lu, Yizhuang Zhang, Jiaguang Li, Jiasheng Wang, Aijun Chen, Fenfen Wen, Ming Shao","doi":"10.21769/BioProtoc.5092","DOIUrl":"https://doi.org/10.21769/BioProtoc.5092","url":null,"abstract":"<p><p>Maternal mRNAs and proteins are produced during oogenesis by more than 60% of zebrafish genes. They are indispensable for fertilization and early embryogenesis. Generation and analysis of the maternal mutant is the most direct way to characterize the maternal function of the specific gene. However, due to the lethality of zygotic mutants, the maternal function of most genes in zebrafish remains elusive. Several methods have been developed to circumvent this obstacle, including mRNA rescue, germ-line replacement, oocyte microinjection in situ, mosaic mutation, and bacterial artificial chromosome (BAC)-mediated conditional rescue. Here, we provide an alternative approach to generate zebrafish maternal mutants rapidly and efficiently by introducing four tandem sgRNA expression cassettes into Tg(<i>zpc:zcas9</i>) embryos. This method is more technically feasible and cost- and time-effective than other established methods. Key features • This protocol can circumvent the lethality or infertility of the zygotic mutants to obtain maternal mutants of the target gene. • This protocol is time-saving (one fish generation). • Using this protocol, double-gene maternal mutants can be obtained in a single generation. • Stable lines can be established to continuously produce maternal mutant embryos for the gene of interest.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"14 21","pages":"e5092"},"PeriodicalIF":1.0,"publicationDate":"2024-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11543609/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142635778","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Semi-Automated Assessment of Long-Term Olfactory Habituation in <i>Drosophila melanogaster</i> Using the Olfactory Arena.","authors":"Camilla Roselli, Mani Ramaswami, Marcia M Aranha","doi":"10.21769/BioProtoc.5102","DOIUrl":"https://doi.org/10.21769/BioProtoc.5102","url":null,"abstract":"<p><p>Long-lasting memories are a core aspect of an animal's life. Such memories are characterized by unique molecular mechanisms and often unique circuitry, neither of which are completely understood in vivo. The deep knowledge of the identity and connectivity of neurons of the fruit fly <i>Drosophila melanogaster</i>, as well as the sophisticated genetic tools that allow in vivo perturbations and physiology monitoring, make it a remarkably useful organism in which to investigate the molecular mechanisms of long-term memories. In this protocol, we focus on habituation, a non-associative form of learning, and describe a reliable, semi-automated technique to induce and assess long-term olfactory habituation (LTH) in <i>Drosophila</i> using the olfactory arena, thus providing a method aligned with recent technological progress in behavioral measurement. Prior work has shown that LTH is induced by a 4-day exposure to an odorant and is characterized by a long-lasting (> 24 h) reduction in behavioral response to the exposed odorant, measured using a manual and skill-intensive Y-maze assay. Here, we present a semi-automated protocol for obtaining quantifiable measures of LTH, at the level of detail required for other investigators in the field. Unlike previously described methods, the protocol presented here provides quantitative and detailed behavioral measurements obtained by video recording that can be shared with the scientific community and allows sophisticated forms of offline analysis. We suggest that this procedure has the potential to advance our understanding of molecular and circuit mechanisms of olfactory habituation, its control via neuromodulation, and its interactions with other forms of memory. Key features • A protocol to induce olfactory aversive long-term habituation in <i>Drosophila melanogaster</i>. • Video recording and analysis of <i>Drosophila</i> in an olfactory arena.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"14 21","pages":"e5102"},"PeriodicalIF":1.0,"publicationDate":"2024-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11543785/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142634315","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Improving Stability of <i>Spiroplasma citri</i> MreB5 Through Purification Optimization and Structural Insights.","authors":"Vani Pande, Pananghat Gayathri","doi":"10.21769/BioProtoc.5086","DOIUrl":"https://doi.org/10.21769/BioProtoc.5086","url":null,"abstract":"<p><p>MreB is a prokaryotic actin homolog. It is essential for cell shape in the majority of rod-shaped cell-walled bacteria. Structural and functional characterization of MreB protein is important to understand the mechanism of ATP-dependent filament dynamics and membrane interaction. In vitro studies on MreBs have been limited due to the difficulty in purifying the homogenous monomeric protein. We have purified MreB from the cell-wall-less bacteria <i>Spiroplasma citri</i>, ScMreB5, using heterologous expression in Escherichia coli. This protocol provides a detailed description of purification condition optimization that led us to obtain high concentrations of stable ScMreB5. Additionally, we have provided a protocol for detecting the presence of monovalent ions in the ScMreB5 AMP-PNP-bound crystal structure. This protocol can be used to obtain a high yield of ScMreB5 for carrying out biochemical and reconstitution studies. The strategies used for ScMreB5 show how optimizing buffer components can enhance the yield and stability of purified protein. Key features • The protocol is a useful approach to standardize purification of nucleotide-dependent cytoskeletal filaments and other nucleotide-binding proteins. • The mechanistic basis of how different ions could stabilize a protein, and hence improve yield in purification, has been demonstrated. • The change in buffer conditions/salt enabled us to get sufficient yield for biochemical and structural characterization.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"14 20","pages":"e5086"},"PeriodicalIF":1.0,"publicationDate":"2024-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11540049/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142607606","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of the Mammary Gland in Mouse: A Whole-Mount Microscopic Analysis.","authors":"Bo Wang, Yuchen Xie, Zejian Yang, Jingyue Zhang, Huiwen Zhang, Peijun Liu","doi":"10.21769/BioProtoc.5089","DOIUrl":"https://doi.org/10.21769/BioProtoc.5089","url":null,"abstract":"<p><p>The mammary gland undergoes functional, developmental, and structural changes that are essential for lactation and reproductive processes. An overview of such unique tissue can offer clearer insights into mammary development and tumorigenesis. Compared to traditional methods, mouse mammary gland whole mount is a pivotal technique that provides three-dimensional structural perspectives on gland morphology and developmental stages, offering an inexpensive and accessible approach. This protocol outlines the tissue isolation of the mouse mammary gland and provides detailed instructions for whole-mount staining and analysis. Mammary gland tissues are carefully dissected from euthanized mice and stained with Carmine Alum to highlight the ductal structures, enabling detailed visualization of the branching patterns and morphological changes. Light microscopy is used to capture a panoramic image of the stained mammary gland, enabling the quantitative analysis of terminal end buds (TEBs) and bifurcated TEBs to further investigate mammary gland remodeling. This method can provide invaluable insights, particularly in the study of mammary gland morphogenesis and tumorigenesis, underscoring its significance in both basic research and clinical applications. Key features • Monitor mammary gland development within 2 days using whole-mount staining.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"14 20","pages":"e5089"},"PeriodicalIF":1.0,"publicationDate":"2024-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11540045/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142607585","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Generation and Maintenance of Patient-Derived Endometrial Cancer Organoids.","authors":"Mali Barbi, Divya Gowthaman, Arielle Katcher, Megan Gorman, Brian Yueh, Aaron Nizam, Charlie Chung, Erdogan Oguzhan Akyildiz, Marina Frimer, Gary L Goldberg, Semir Beyaz","doi":"10.21769/BioProtoc.5093","DOIUrl":"https://doi.org/10.21769/BioProtoc.5093","url":null,"abstract":"<p><p>Endometrial cancer (EC) is the leading cause of gynecologic cancer morbidity and mortality in the U.S. Despite advancements in cancer research, EC death rates are increasing, particularly high-grade endometrial cancers. The development of three-dimensional (3D) patient-derived organoid (PDO) models for EC is crucial, as they provide a more accurate representation of the biological and genetic complexity of a patient's tumor compared to traditional 2D cell lines. Here, we describe a protocol for cultivating PDO models from normal endometrium and EC across different EC subtypes. These EC PDO models can be expanded across multiple passages and facilitate the exploration of tumor behavior and drug responses, thereby advancing our understanding of the disease and potentially leading to more effective and individualized novel therapeutic strategies. Key features • Establishment of patient-derived EC and normal endometrium organoids. • Accurate replication of the various histologic and molecular subtypes of EC within the organoids. • Our approach provides a clinically relevant platform for studying EC development, metastasis, progression, and recurrence. • It offers potential for developing targeted therapeutic interventions tailored to specific EC subtypes. Graphical overview Schematic overview of endometrial cancer and normal organoid preparation from patient-derived samples.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"14 20","pages":"e5093"},"PeriodicalIF":1.0,"publicationDate":"2024-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11539961/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142607598","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Host Receptor Pili for Cryo-EM Single-Particle Reconstruction.","authors":"Ran Meng","doi":"10.21769/BioProtoc.5094","DOIUrl":"https://doi.org/10.21769/BioProtoc.5094","url":null,"abstract":"<p><p>Single-stranded RNA bacteriophages (ssRNA phages) infect their hosts by binding to the host receptor pili. Purification of pili usually involves mechanical shearing of pili from cells followed by precipitation. However, previous methods often result in low efficiency or unstable results due to pili retraction. This protocol presents an optimized method for purifying receptor type IV pili from <i>Acinetobacter genomospecies 16</i> (<i>A. gp16</i>), incorporating enhancements in shearing and collection steps to achieve high yields. We found that repeated passage through syringe needles increases yield, and temperature control is crucial during purification. Additionally, the CsCl density gradient was optimized specifically for this specific strain. The purified type IV pili are suitable for cryogenic electron microscopy (cryo-EM) and various biochemical experiments. Key features • Pili purification for single-particle cryo-electron microscopy (Cryo-EM) analysis • This protocol builds upon the F-pili purification method developed by Costa et al. [1] extending its application to the <i>Acinetobacter genomosp.</i> 16. • It is optimized for higher and more stable pili yields, as well as increased reproducibility. • The method is tested on various bacterial species and can be adapted to purify different types of pili.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"14 20","pages":"e5094"},"PeriodicalIF":1.0,"publicationDate":"2024-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11539960/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142607601","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An Improved Focus-Forming Assay for Determination of the Dengue Virus Titer.","authors":"Maharah Binte Abdul Mahid, Pradeep Bist, Kristmundur Sigmundsson, Muhammad Danial Bin Mohd Mazlan, Satoru Watanabe, Milly M Choy, Subhash G Vasudevan, Kitti Wing Ki Chan","doi":"10.21769/BioProtoc.5084","DOIUrl":"10.21769/BioProtoc.5084","url":null,"abstract":"<p><p>Dengue virus (DENV), a common and prevalent mosquito-borne endemic disease, is caused by four serotypes (DENV-1-4) and has spread rapidly on a global scale over the past decade. A crucial step in the development of antiviral therapeutics requires the utilization of in vitro cell-based techniques, such as plaque assays and focus-forming assays (FFA) for virus quantification. Vero cells have been widely used for FFA and plaque assay; however, there are instances when their efficacy and efficiency in the detection of certain clinical DENV isolates are low. Here, we showed that BHK-21 cells are more sensitive than Vero cells in the detection of all DENV-1-4 plaques and foci. In addition, we developed an improved FFA protocol for the quantification of all four DENV serotypes. Using a pan-flavivirus envelope (E) antibody, we reduce the possibility of false positives by defining a focus to consist of a minimum of eight infected cells. We outlined a protocol using the Operetta® high-content imaging system to automate the digital capture of these infected cells. A pipeline was also designed using the CellProfilerTM automated image analysis software to detect these foci. We then compare the results of the improved FFA with plaque assay. Notably, the improved FFA detected clear foci of the DENV-4 strain that does not form distinct plaques. We subsequently demonstrated the potential application of the improved FFA protocol in antiviral testing, utilizing a nucleoside inhibitor of DENV, NITD008 as a control. The protocol is amenable to a diverse array of applications, including high-throughput compound screening (HTS). Key features • An improved focus-forming assay that has the potential to quantify the DENV-4 strain, which was previously hard to plaque. • Improvements have been made to reduce the possibility of false positives. • Improved workflow using automated digital imaging process and counting of foci. • Applicable to antiviral compounds screening and is amenable to high-throughput screening.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"14 20","pages":"e5084"},"PeriodicalIF":1.0,"publicationDate":"2024-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11557373/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142635765","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Detecting Native Protein-Protein Interactions by APEX2 Proximity Labeling in <i>Drosophila</i> Tissues.","authors":"Jhen-Wei Wu, Chueh-Wen Wang, Wei Yang Hong, Anna C C Jang, Yu-Chiuan Chang","doi":"10.21769/BioProtoc.5090","DOIUrl":"https://doi.org/10.21769/BioProtoc.5090","url":null,"abstract":"<p><p>Enzyme-catalyzed proximity labeling is a potent technique for the discernment of subtle molecular interactions and subcellular localization, furnishing contextual insights into the protein of interest within cells. Although ascorbate peroxidase2 (APEX2) has proven effective in this approach when overexpressed, its compatibility with endogenous proteins remains untested. We improved this technique for studying native protein-protein interactions in live <i>Drosophila</i> ovary tissue. Through CRISPR/Cas9 genome editing, APEX2 was fused with the endogenous dysfusion gene. By pre-treating the tissue with Triton X-100 to enhance biotin-phenol penetration, we successfully identified multiple proteins interacting with the native Dysfusion proteins that reside on the inner nuclear membrane. Our protocol offers a comprehensive workflow for delineating the interactome networks of ovarian components in <i>Drosophila</i>, aiding future studies on endogenous protein-protein interactions in various tissues of other animals. Key features • Elucidating Protein-protein interactions provides deeper insights into the regulation of gene expression in molecular network and complex signaling pathways, advancing protein engineering and drug design. • This protocol utilizes CRISPR/Cas9 knock-in technology to tag endogenous proteins with the APEX2 to label nearby proteins within a 20 nm radius in <i>Drosophila melanogaster</i>. • We optimize APEX2-proximity labeling by using Triton X-100 pre-treatment to enhance biotin-phenol penetration into <i>Drosophila</i> ovaries, enabling endogenous proteins enrichment under native conditions.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"14 20","pages":"e5090"},"PeriodicalIF":1.0,"publicationDate":"2024-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11540050/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142607568","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Chemogenetic Silencing of Neonatal Spontaneous Activity of Projection Neurons in the Dorsal Striatum of Mice.","authors":"Bojana Kokinovic, Maria Ryazantseva, Svetlana Molchanova","doi":"10.21769/BioProtoc.5088","DOIUrl":"https://doi.org/10.21769/BioProtoc.5088","url":null,"abstract":"<p><p>Neuroscience incorporates manipulating neuronal circuitry to enhance the understanding of intricate brain functions. An effective strategy to attain this objective entails utilizing viral vectors to induce varied gene expression by delivering transgenes into brain cells. Here, we combine the use of transgenic mice, neonatal transduction with adeno-associated viral constructs harboring inhibitory designer receptor exclusively activated by designer drug (DREADD) gene, and the DREADD agonist clozapine N-oxide (CNO). In this way, a chemogenetic approach is employed to suppress neuronal activity in the region of interest during a critical developmental window, with subsequent investigation into its effects on the neuronal circuitry in adulthood. Key features • Comprehensive protocol for newborn viral transduction in the dorsal striatum of mice • Uses a viral construct encoding inhibitory DREADD under the control of Cre recombinase to attenuate the activity of specific cell types in the brain.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"14 20","pages":"e5088"},"PeriodicalIF":1.0,"publicationDate":"2024-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11539959/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142607436","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}