往期刊物2017
卷册: 7, 期号: 11
生物化学
Heavy Metal Stress Assay of Caenorhabditis elegans
秀丽隐杆线虫重金属胁迫检测
Organisms have developed many protective systems to reduce the toxicity from heavy metals. The nematode Caenorhabditis elegans has been widely used to determine the protective mechanisms against heavy metals. Responses against heavy metals can be monitored by expression of reporter genes, while sensitivity can be determined by quantifying growth or survival rate following exposure to heavy metals.
癌症生物学
DNA Fiber Assay upon Treatment with Ultraviolet Radiations
紫外线辐射处理后DNA纤维的检测
Genome stability is continuously challenged by a wide range of DNA damaging factors. To promote a correct DNA repair and cell survival, cells orchestrate a coordinated and finely tuned cascade of events collectively known as the DNA Damage Response (DDR). Ultra Violet (UV) rays are among the main environmental sources of DNA damage and a well recognized cancer risk factor. UV rays induce the formation of toxic cyclobutane-type pyrimidine dimers (CPD) and [6-4]pyrimidine-pyrimidone (6-4PP) photoproducts which trigger the activation of the intra-S phase cell cycle checkpoint (Kaufmann, 2010) aimed at preventing replication fork collapse, late origin firing, and stabilizing fragile sites (Branzei and Foiani, 2009). To monitor the activation of the intra-S phase checkpoint in response to UV type C (UVC) exposure, the DNA fiber assay can be used to analyse the new origin firing and DNA synthesis rate (Jackson et al., 1998; Merrick et al., 2004; Alfano et al., 2016). The DNA fiber assay technique was conceived in the 90s and then further developed through the use of thymidine analogues (such as CldU and IdU), which are incorporated into the nascent DNA strands. By treating the cells in sequential mode with these analogues, which can be visualized through specific antibodies carrying different fluorophores, it is possible to monitor the replication fork activity and assess how this is influenced by UV radiations or others agents.
Targeted Nucleotide Substitution in Mammalian Cell by Target-AID
在哺乳动物细胞中通过靶向性AID进行核苷酸靶向置换
Programmable RNA-guided nucleases based on CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) systems have been applied to various type of cells as powerful genome editing tools. By using activation-induced cytidine deaminase (AID) in place of the nuclease activity of the CRISPR/Cas9 system, we have developed a genome editing tool for targeted nucleotide substitution (C to T or G to A) without donor DNA template (Figure 1; Nishida et al., 2016). Here we describe the detailed method for Target-AID to perform programmable point mutagenesis in the genome of mammalian cells. A specific method for targeting the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in Chinese Hamster Ovary (CHO) cell was described here as an example, while this method principally should be applicable to any gene of interest in a wide range of cell types.Figure 1. Schematic illustration for Target-AID and its targetable site. In a guide-RNA (gRNA)-dependent manner, PmCDA1 fused to nCas9 (D10A) via a linker performs programmable cytidine mutagenesis around -21 to -16 positions relative to PAM sequence on the non-complementary strand in mammalian cells. The targetable site was determined based on the efficient base substitution (> 20%) observed in the previous work.
Single-molecule Analysis of DNA Replication Dynamics in Budding Yeast and Human Cells by DNA Combing
采用DNA梳理技术对芽殖酵母和人类细胞中DNA复制动力学进行单分子分析
The DNA combing method allows the analysis of DNA replication at the level of individual DNA molecules stretched along silane-coated glass coverslips. Before DNA extraction, ongoing DNA synthesis is labeled with halogenated analogues of thymidine. Replication tracks are visualized by immunofluorescence using specific antibodies. Unlike biochemical and NGS-based methods, DNA combing provides unique information on cell-to-cell variations in DNA replication profiles, including initiation and elongation. Finally, this assay can be used to monitor the effect of DNA lesions on fork progression, arrest and restart.
Intracaecal Orthotopic Colorectal Cancer Xenograft Mouse Model
脑内原位结直肠癌异种移植小鼠模型
The host microenvironment plays a prominent role in tumor growth, angiogenesis, invasion, metastasis, and response to therapy. Orthotopic tumor model mimics the natural environment of tumor development and provides an effective approach to investigate tumor pathophysiology and develop therapeutic strategies. This protocol describes the technique involving injection of colorectal cancer cell suspension into the intestinal wall of mice to establish an orthotopic colorectal tumor model.
Fluorometric Estimation of Glutathione in Cultured Microglial Cell Lysate
培养的小胶质细胞裂解液中谷胱甘肽的荧光法估测
Glutathione is one of the major antioxidant defense components present in cells. It is predominantly present as reduced glutathione (GSH) and converted into oxidized glutathione (GSSG) while reducing the free radicals like hydroxyl ions (OH-). For the measurement of GSH and GSSG, o-phthalaldehyde (OPT) has been used as a fluorescent reagent. O-phthalaldehyde has an ability to react specifically with GSH at pH 8 and GSSG at pH 12 respectively. N-ethylmaleimide (NEM) has been used to prevent auto-oxidation of GSH during measurement of GSSG in the present protocol. The original protocol by Hissin and Hilf was developed for glutathione estimation in Rat liver tissue. The present protocol has been standardized following Hissin and Hilf (1976) for the estimation of glutathione in cultured microglial cell lysate but it can also be used for other mammalian cell lysate. In our lab same protocol has been used for the estimation of glutathione in the whole cell lysate of murine neuroblastoma cell, N2a.
Pituitary Isograft Transplantation in Mice
小鼠垂体同系移植物移植
The mouse pituitary isograft is a technique developed to administer persistent hormone stimulation, thereby increasing cellular proliferation in the mammary tissue (Christov et al., 1993). The pituitary isograft procedure was first described in ‘Induction of Mammary Cancer in Mice without the Mammary Tumor Agent by Isografts of Hypophyses’ by O. Mühlbock and L. M. Boot in 1959 (Muhlbock and Boot, 1959). Since then, the procedure has seen wide use. A pituitary gland is harvested posthumously from a donor mouse and implanted under the renal capsule of the recipient mouse through a small abdominal incision just below the last rib. Once the pituitary gland is implanted, it begins releasing hormones. These secretions increase serum levels of multiple hormones including prolactin, progesterone and 17β-estradiol (Christov et al., 1993). Although the effects of these hormones on cancer cell proliferation, growth, differentiation, and longevity are not well characterized, and, in some cases, controversial, the net effect of a pituitary isograft is to increase the proliferation of murine breast tissue depending upon strain specific characteristics (Lydon et al., 1999).Below is a protocol describing how to perform the pituitary isograft procedure. After many of the steps, a time reference is listed in parentheses. Each reference corresponds to a time point in the embedded video of the procedure. (Video 1) Video 1. Pituitary isograft transplantation in mice. Video portraying pituitary isograft transplantation procedure in donor and recipient mice.
Whole Mammary Gland Transplantation in Mice Protocol
小鼠全乳腺移植方案
Whole Mammary Gland Transplantation involves transplanting an excised mammary gland into another, more suitable host. This method can be used to extend the life of a mammary gland past the mouse’s life span by transplanting the mammary gland of an older mouse into a young healthy mouse. As you can see in the video below (Video 1), by attaching it to the abdomen of the mouse, the gland will receive a steady blood supply and both epithelial and stromal cells will remain viable for up to one year. Although this method is not used often, it has been part of several experiments including determining whether the stroma or epithelium is the primary target in chemically induced mouse mammary tumorigenesis (Medina and Kittrell, 2005). To monitor transplants, palpate every week for tumor formation. The transplanted mammary gland may also be passaged serially every 8-10 weeks. Keep transplanted gland in the same mouse for no longer than one year. Video 1. Whole mammary gland transplantation
细胞生物学
Generation of Mutant Pigs by Direct Pronuclear Microinjection of CRISPR/Cas9 Plasmid Vectors
通过直接原核显微注射CRISPR/Cas9质粒载体生成突变株
A set of Cas9 and single guide CRISPR RNA expression vectors was constructed. Only a very simple procedure was needed to prepare specific single-guide RNA expression vectors with high target accuracy. Since the de novo zygotic transcription had been detected in mouse embryo at the 1-cell stage, the plasmid DNA vectors encoding Cas9 and GGTA1 gene specific single-guide RNAs were micro-injected into zygotic pronuclei to confirm such phenomenon in 1-cell pig embryo. Our results demonstrated that mutations caused by these CRISPR/Cas9 plasmids occurred before and at the 2-cell stage of pig embryos, indicating that besides the cytoplasmic microinjection of in vitro transcribed RNA, the pronuclear microinjection of CRISPR/Cas9 DNA vectors provided an efficient solution to generate gene-knockout pig.
Flow Cytometric Analysis of HIV-1 Transcriptional Activity in Response to shRNA Knockdown in A2 and A72 J-Lat Cell Lines
A2和A72 J-Lat细胞系中HIV-1经shRNA沉默后转录活性的流式细胞分析
The main obstacle to eradicating HIV-1 from patients is post-integration latency (Finzi et al., 1999). Antiretroviral treatments target only actively replicating virus, while latent infections that have low or no transcriptional activity remain untreated (Sedaghat et al., 2007). To eliminate viral reservoirs, one strategy focuses on reversing HIV-1 latency via ‘shock and kill’ (Deeks, 2012). The basis of this strategy is to overcome the molecular mechanisms of HIV-1 latency by therapeutically inducing viral gene and protein expression under antiretroviral therapy and to cause selective cell death via the lytic properties of the virus, or the immune system now recognizing the infected cells. Recently, a number of studies have described the therapeutic potential of pharmacologically inhibiting members of the bromodomain and extraterminal (BET) family of human bromodomain proteins (Filippakopoulos et al., 2010; Dawson et al., 2011; Delmore et al., 2011) that include BRD2, BRB3, BRD4 and BRDT. Small-molecule BET inhibitors, such as JQ1 (Filippakopoulos et al., 2010; Delmore et al., 2011), I-BET (Nicodeme et al., 2010), I-Bet151 (Dawson et al., 2011), and MS417 (Zhang et al., 2012) successfully activate HIV transcription and reverse viral latency in clonal cell lines and certain primary T-cell models of latency. To identify the mechanism by which BET proteins regulate HIV-1 latency, we utilized small hairpin RNAs (shRNAs) that target BRD2, BRD4 and Cyclin T1, which is a component of the critical HIV-1 cofactor positive transcription elongation factor b (P-TEFb) and interacts with BRD2, and tested them in the CD4+ J-Lat A2 and A72 cell lines. The following protocol describes a flow cytometry-based method to determine the amount of transcriptional activation of the HIV-1 LTR upon shRNA knockdown. This protocol is optimized for studying latently HIV-1-infected Jurkat (J-Lat) cell lines.
免疫学
ELISPOT Assay to Measure Peptide-specific IFN-γ Production
采用ELISPOT测定法测定肽特异性IFN-γ的生成
Interferon-gamma (IFN-γ) is crucial for immunity against intracellular pathogens and for tumor control. It is produced predominantly by natural killer (NK) and natural killer T cells (NKT) as well as by antigen-specific Th1 CD4+ and CD8+ effector T cells. When investigating immune responses against pathogens and cancer cells, measuring antigen-specific cytokine-responses by cells of adaptive immunity offers an advantage over total non-specific cytokine responses. Significantly, the measurement of antigen-specific IFN-γ responses against pathogens or cancer cells, when compared to a treatment group, provides a quantitative measure of how well the treatment works. Measuring antigen-specific IFN-γ responses involves culture of the cells being considered (CD4+ or CD8+ T cells) with antigen presenting cells (APC) and a specific peptide from the target pathogen or cancer cell compared to control cultures without a peptide. After a suitable timeframe, the cytokine released is measured by an ELISPOT assay. The difference in the number of cells secreting IFN-γ, with and without peptide, is a measure of antigen-specific IFN-γ responses. This assay can be applied to other cytokines such as IL-10.
In vitro Antigen-presentation Assay for Self- and Microbial-derived Antigens
自身抗原和微生物源性抗原的体外抗原呈递测定
Antigen presenting cells (APC) are able to process and present to T cells antigens from different origins. This mechanism is highly regulated, in particular by Patter Recognition Receptor (PRR) signals. Here, I detail a protocol designed to assess in vitro the capacity of APC to present antigens derived from bacteria, apoptotic and infected apoptotic cells.
Isolation and Infection of Drosophila Primary Hemocytes
果蝇原代血细胞的分离和感染
Phagocytosis of invading pathogens and their subsequent clearance in lysosomes is important for organismal fitness. We have devised the following protocol to extract phagocytic hemocytes from wild-type and mutant Drosophila larvae and infect the isolated hemocytes with GFP-labeled E. coli to measure the rate of phagocytosis and degradation within individual hemocytes over time.
微生物学
Expression and Purification of the Cas10-Csm Complex from Staphylococci
表达并纯化葡萄球菌的Cas10-Csm复合体
CRISPR-Cas (Clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) is a class of prokaryotic immune systems that degrade foreign nucleic acids in a sequence-specific manner. These systems rely upon ribonucleoprotein complexes composed of Cas nucleases and small CRISPR RNAs (crRNAs). Staphylococcus epidermidis and Staphylococcus aureus are bacterial residents on human skin that are also leading causes of antibiotic resistant infections (Lowy, 1998; National Nosocomial Infections Surveillance, 2004; Otto, 2009). Many staphylococci possess Type III-A CRISPR-Cas systems (Marraffini and Sontheimer, 2008; Cao et al., 2016), which have been shown to prevent plasmid transfer and protect against viral predators (Goldberg et al., 2014; Hatoum-Aslan et al., 2014; Samai et al., 2015) in these organisms. Thus, gaining a mechanistic understanding of these systems in the native staphylococcal background can lead to important insights into the factors that impact the evolution and survival of these pathogens. Type III-A CRISPR-Cas systems encode a five-subunit effector complex called Cas10-Csm (Hatoum-Aslan et al., 2013). Here, we describe a protocol for the expression and purification of Cas10-Csm from its native S. epidermidis background or a heterologous S. aureus background. The method consists of a two-step purification protocol involving Ni2+-affinity chromatography and a DNA affinity biotin pull-down, which together yield a pure preparation of the Cas10-Csm complex. This approach has been used previously to analyze the effects of mutations on Cas10-Csm complex integrity (Hatoum-Aslan et al., 2014), crRNA formation (Hatoum-Aslan et al., 2013), and to detect binding partners that directly interact with the core Cas10-Csm complex (Walker et al., 2016). Importantly, this approach can be easily adapted for use in other Staphylococcus species to probe and understand their native Type III-A CRISPR-Cas systems.
A Reliable Assay to Evaluate the Virulence of Aspergillus nidulans Using the Alternative Animal Model Galleria mellonella (Lepidoptera)
以大蜡螟（鳞翅目）为动物替代模型评估构巢曲霉毒力的可靠实验方法
The greater wax moth Galleria mellonella has emerged as an effective heterologous host to study fungal pathogenesis and the efficacy of promising antifungal drugs (Mylonakis et al., 2005; Li et al., 2013). Here, a methodology describing the Aspergillus nidulans infection in G. mellonella larvae, along with insect survival analysis, is reported. This protocol allowed the distinction between virulent A. nidulans strains (such as TNO2A3), which induced high larval mortality rates, to those in which gene deletion was accompanied by reduced pathogenicity such as ∆gcsA and ∆sdeA (Fernandes et al., 2016).
Fluorescently Labelled Aerolysin (FLAER) Labelling of Candida albicans Cells
使用荧光标记的嗜水气单胞菌溶素变异体（FLAER）标记白色念珠菌细胞
In this protocol we describe a nonradiolabelled labelling of GPI anchor in Candida albicans. The method uses a fluorescent probe to bind specifically to GPI anchors so that the level of GPI-anchored proteins at the cell surface can be measured. The labelling does not need permeabilization of cells and can be carried out in vivo.
Phototaxis Assays of Synechocystis sp. PCC 6803 at Macroscopic and Microscopic Scales
宏观和微观尺度上集胞藻PCC 6803的趋光性分析
Phototaxis is a mechanism that allows cyanobacteria to respond to fluctuations in the quality and quantity of illumination by moving either towards or away from a light source. Phototactic movement on low concentration agar or agarose plates can be analyzed at macroscopic and microscopic scales representing group behavior and single cell motility, respectively. Here, we describe a detailed procedure for phototaxis assays on both scales using the unicellular cyanobacterium Synechocystis sp. PCC 6803.
Induction and Quantification of Patulin Production in Penicillium Species
青霉菌展青霉素生成的诱导和定量
Patulin, a worldwide regulated mycotoxin, is primarily produced by Penicillium and Aspergillus species during fruit spoilage. Patulin contamination is a great concern with regard to human health because exposure of the mycotoxin can result in severe acute and chronic toxicity, including neurotoxic, mutagenic, and immunotoxic effects. Penicillium expansum is known as the main producer of patulin. This protocol addresses the cultivation procedure of P. expansum under patulin permissive conditions and describes the method of collection and detection of patulin.
Protein Localization in the Cyanobacterium Anabaena sp. PCC7120 Using Immunofluorescence Labeling
免疫荧光标记定位蓝藻鱼腥藻属PCC7120中的蛋白质
Techniques such as immunoflorescence are widely used to determine subcellular distribution of proteins. Here we report on a method to immunolocalize proteins in Anabaena sp. PCC7120 with fluorophore-conjugated antibodies by fluorescence microscopy. This method improves the permeabilization of cyanobacterial cells and minimizes the background fluorescence for non-specific attachments. In this protocol, rabbit antibodies were raised against the synthetic peptide of CyDiv protein (Mandakovic et al., 2016). The secondary antibody conjugated to the fluorophore Alexa488 was used due to its different emission range in comparison to the autofluorescence of the cyanobacterium.
Ex vivo Model of Human Aortic Valve Bacterial Colonization
人类主动脉瓣膜细菌定植离体模型
The interaction of pathogens with host tissues is a key step towards successful colonization and establishment of an infection. During bacteremia, pathogens can virtually reach all organs in the human body (e.g., heart, kidney, spleen) but host immunity, blood flow and tissue integrity generally prevents bacterial colonization. Yet, patients with cardiac conditions (e.g., congenital heart disease, atherosclerosis, calcific aortic stenosis, prosthetic valve recipients) are at a higher risk of bacterial infection. This protocol was adapted from an established ex vivo porcine heart adhesion model and takes advantage of the availability of heart tissues obtained from patients that underwent aortic valve replacement surgery. In this protocol, fresh tissues are used to assess the direct interaction of bacterial pathogens associated with cardiovascular infections, such as the oral bacterium Streptococcus mutans, with human aortic valve tissues.
分子生物学
Chromatin Immunoprecipitation Experiments from Whole Drosophila Embryos or Larval Imaginal Discs
整个果蝇胚胎或幼虫成虫盘的染色质免疫沉淀实验
Chromatin Immunoprecipitation coupled either to qPCR (qChIP) or high-throughput sequencing (ChIP-Seq) has been extensively used in the last decades to identify the DNA binding sites of transcription factors or the localization of various histone marks along the genome. The ChIP experiment generally includes 7 steps: collection of biological samples (A), cross-linking proteins to DNA (B), chromatin isolation and fragmentation by sonication (C), sonication test (D), immunoprecipitation with antibodies against the protein or the histone mark of interest (E), DNA recovery (E), identification of factor-associated DNA sequences by PCR or sequencing (F). The protocol described here can readily be used for ChIP-seq and ChIP-qPCR experiments. The entire procedure, describing experimental setup conditions to optimize assays in intact Drosophila tissues, can be completed within four days.
A Protocol for Production of Mutant Mice Using Chemically Synthesized crRNA/tracrRNA with Cas9 Nickase and FokI-dCas9
利用化学合成的crRNA/tracrRNA-Cas9 Nick酶及FokI-dCas9产生突变小鼠的实验方法
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is the most widely used genome editing tool. A common CRISPR/Cas9 system consists of two components: a single-guide RNA (sgRNA) and Cas9. Both components are required for the introduction of a double-strand break (DSB) at a specific target sequence. One drawback of this system is that the production of sgRNA in the laboratory is laborious since it requires cloning of an sgRNA sequence, in vitro transcription reaction and sgRNA purification. An alternative to targeting Cas9 activity by sgRNA is to target it with two small RNAs: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). Both of these small RNAs can be chemically synthesized which makes the production of these RNAs less difficult when compared to sgRNA. Another downside of the CRISPR/Cas9 systems is that off-target effects have been reported. However, modified forms of Cas9 have been developed to minimize off-target effects. For example, nickase-type Cas9 (nCas9) and FokI domain-fused catalytically-inactive Cas9 (FokI-dCas9; fCas9) induce DSBs only when two guide RNAs bind opposite strands within a defined distance. In this protocol, we describe our experimental system for the production of mutant mice using a CRISPR/Cas9 system that combines crRNA, tracrRNA, and modified forms of Cas9. This method not only facilitates the preparation of reagents for the genome editing system but it can also reduce the risk of off-target effects.
Formation of Minimised Hairpin Template-transcribing Dumbbell Vectors for Small RNA Expression
用于小分子RNA表达的最小化发夹模板转录哑铃形载体生成
A major barrier for using non-viral vectors for gene therapy is the short duration of transgene expression in postmitotic tissues. Previous studies showed transgene expression from conventional plasmid fell to sub-therapeutic level shortly after delivery even though the vector DNA was retained, suggesting transcription was silenced in vivo (Nicol et al., 2002; Chen et al., 2004). Emerging evidence indicates that plasmid bacterial backbone sequences are responsible for the transcriptional repression and this process is independent of CpG methylation (Chen et al., 2008). Dumbbell-shaped DNA vectors consisting solely of essential elements for transgene expression have been developed to circumvent these drawbacks. This novel non-viral vector has been shown to improve transgene expression in vitro and in vivo (Schakowski et al., 2001 and 2007). Here we describe a novel method for fast and efficient production of minimised small RNA-expressing dumbbell vectors. In brief, the PCR-amplified promoter sequence is ligated to a chemically synthesized hairpin RNA coding DNA template to form the covalently closed dumbbell vector. This new technique may facilitate applications of dumbbell-shaped vectors for preclinical investigation and human gene therapy.
神经科学
Kinetic Lactate Dehydrogenase Assay for Detection of Cell Damage in Primary Neuronal Cell Cultures
采用乳酸脱氢酶动力测定法检测原代神经元细胞培养物中的细胞损伤
The aim of many in vitro models of acute or chronic degenerative disorders in the neurobiology field is the assessment of survival or damage of neuronal cells. Damage of cells is associated with loss of outer cell membrane integrity and leakage of cytoplasmic cellular proteins. Therefore, activity assays of cytoplasmic enzymes in supernatants of cell cultures serve as a practicable tool for quantification of cellular injury (Koh and Choi, 1987; Bruer et al., 1997). Lactate dehydrogenase (LDH) is such a ubiquitously expressed cytosolic enzyme, which is very stable due to a very long protein half-life (Hsieh and Blumenthal, 1956; Koh and Cotman, 1992; Koh et al., 1995).
The Repeated Flurothyl Seizure Model in Mice
三氟乙醚诱导的小鼠反复癫痫发作模型
Development of spontaneous seizures is the hallmark of human epilepsy. There is a critical need for new epilepsy models in order to elucidate mechanisms responsible for leading to the development of spontaneous seizures and for testing new anti-epileptic compounds. Moreover, rodent models of epilepsy have clearly demonstrated that there are two independent seizure systems in the brain: 1) the forebrain seizure network required for the expression of clonic seizures mediated by forebrain neurocircuitry, and 2) the brainstem seizure network necessary for the expression of brainstem or tonic seizures mediated by brainstem neurocircuitry. In seizure naïve animals, these two systems are separate, but developing models that can explore the intersection of the forebrain and brainstem seizure systems or for elucidating mechanisms responsible for bringing these two seizure systems together may aid in our understanding of: 1) how seizures can become more complex over time, and 2) sudden unexpected death in epilepsy (SUDEP) since propagation of seizure discharge from the forebrain seizure system to the brainstem seizure system may have an important role in SUDEP because many cardiorespiratory systems are localized in the brainstem. The repeated flurothyl seizure model of epileptogenesis, as described here, may aid in providing insight into these important epilepsy issues in addition to understanding how spontaneous seizures develop.
植物科学
DNA-free Genome Editing of Chlamydomonas reinhardtii Using CRISPR and Subsequent Mutant Analysis
利用CRISPR技术对莱茵衣藻进行DNA-free的基因组编并对产生突变进行分析
We successfully introduced targeted knock-out of gene of interest in Chlamydomonas reinhardtii by using DNA-free CRISPR. In this protocol, the detailed procedures of an entire workflow cover from the initial target selection of CRISPR to the mutant analysis using next generation sequencing (NGS) technology. Furthermore, we introduce a web-based set of tools, named CRISPR RGEN tools (http://www.rgenome.net/), which provides all required tools from CRISPR target design to NGS data analysis.
Determining Genome Size from Spores of Seedless Vascular Plants
无种子维管束植物孢子基因组大小的测定
Seedless vascular plants, including ferns and lycophytes, produce spores to initiate the gametophyte stage and to complete sexual reproduction. Approximately 10% of them are apomictic through the production of genomic unreduced spores. Being able to measure the spore nuclear DNA content is therefore important to infer their reproduction mode. Here we present a protocol of spore flow cytometry that allows an efficient determination of the reproductive modes of seedless vascular plants.
Protocol for Enrichment of the Membrane Proteome of Mature Tomato Pollen
成熟番茄花粉膜蛋白质组富集实验
We established and elaborated on a method to enrich the membrane proteome of mature pollen from economically relevant crop using the example of Solanum lycopersicum (tomato). To isolate the pollen protein fraction enriched in membrane proteins, a high salt concentration (750 mM of sodium chloride) was used. The membrane protein-enriched fraction was then subjected to shotgun proteomics for identification of proteins, followed by in silico analysis to annotate and classify the detected proteins.
Photometric Assays for Chloroplast Movement Responses to Blue Light
光度法测定叶绿体对蓝光的运动反应
Assessment of chloroplast movements by measuring changes in leaf transmittance is discussed, with special reference to the conditions necessary for reliable estimation of blue light–activated chloroplast responses.
Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides
拟南芥粘液多糖的全种子免疫标记
In addition to synthesizing and secreting copious amounts of pectic polymers (Young et al., 2008), Arabidopsis thaliana seed coat epidermal cells produce small amounts of cellulose and hemicelluloses typical of secondary cell walls (Voiniciuc et al., 2015c). These components are intricately linked and are released as a large mucilage capsule upon hydration of mature seeds. Alterations in the structure of minor mucilage components can have dramatic effects on the architecture of this gelatinous cell wall. The immunolabeling protocol described here makes it possible to visualize the distribution of specific polysaccharides in the seed mucilage capsule.
干细胞
Functional Analysis of Connexin Channels in Cultured Cells by Neurobiotin Injection and Visualization
通过神经生物素注射和可视化对培养细胞中连接蛋白通道进行功能分析
Functional gap junction channels between neighboring cells can be assessed by microinjection of low molecular weight tracer substances into cultured cells. The extent of direct intercellular communication can be precisely quantified by this method. This protocol describes the iontophoretic injection and visualisation of Neurobiotin into cultured cells.