往期刊物2020
卷册: 10, 期号: 4
生物化学
Photoactivable Cholesterol as a Tool to Study Interaction of Influenza Virus Hemagglutinin with Cholesterol
利用光活化胆固醇研究流感病毒血凝素与胆固醇相互作用
Non-covalent binding of cholesterol to the transmembrane region of proteins affect their functionalities, but methods to prove such an interaction are rare. We describe our protocol to label the hemagglutinin (HA) of Influenza virus with a cholesterol derivative in living cells or with immunoprecipitated protein. We synthesized a “clickable” photocholesterol compound, which closely mimics authentic cholesterol. It contains a reactive diazirine group that can be activated by UV-illumination to form a covalent bond with amino acids in its vicinity. Incorporation of photocholesterol into HA is then visualized by “clicking” it to a fluorophore, which can be detected in an SDS-gel by fluorescence scanning. This method provides a convenient and practical way to demonstrate cholesterol-binding to other proteins and probably to identify the binding site.
细胞生物学
CRISPR-Cas9 Genome Editing of Plasmodium knowlesi
诺氏疟原虫CRISPR-Cas9基因编辑
Plasmodium knowlesi is a zoonotic malaria parasite in Southeast Asia that can cause severe and fatal malaria in humans. The main hosts are Macaques, but modern diagnostic tools reveal increasing numbers of human infections. After P. falciparum, P. knowlesi is the only other malaria parasite capable of being maintained in long term in vitro culture with human red blood cells (RBCs). Its closer ancestry to other non-falciparum human malaria parasites, more balanced AT-content, larger merozoites and higher transfection efficiencies, gives P. knowlesi some key advantages over P. falciparum for the study of malaria parasite cell/molecular biology. Here, we describe the generation of marker-free CRISPR gene-edited P. knowlesi parasites, the fast and scalable production of transfection constructs and analysis of transfection efficiencies. Our protocol allows rapid, reliable and unlimited rounds of genome editing in P. knowlesi requiring only a single recyclable selection marker.
Estimating Cellular Abundances of Halo-tagged Proteins in Live Mammalian Cells by Flow Cytometry
利用流式细胞术进行哺乳动物活细胞中Halo 标记蛋白细胞丰度测定
Accurate abundance measurements of cellular proteins are required to achieve a quantitative and predictive understanding of any biological process inside the cell. Existing methods to determine absolute protein abundances are labor-intensive and/or require sophisticated experimental and computational infrastructure (e.g., fluorescence correlation spectroscopy (FCS)-calibrated imaging and quantitative mass spectrometry). Here we detail a straightforward flow cytometry-based method to measure the absolute abundance of any Halo-tagged protein in live cells that uses a standard mammalian cell line with a known number of Halo-CTCF proteins recently characterized in our lab. The protocol only comprises a few steps. First, a cell line expressing the Halo-tagged protein of interest is grown and labeled side-by-side with our standard line. Then, average fluorescence intensities are measured by conventional flow cytometry analysis and finally a simple calculation is applied to estimate the absolute number of the Halo-tagged protein of interest per cell. Once the protein of interest has been endogenously tagged with HaloTag, which we routinely achieve by Cas9-mediated genome editing, the presented protocol is fast, convenient, reproducible, cost-effective and readily accessible.
Preparation of Single Epithelial Cells Suspension from Mouse Mammary Glands
小鼠乳腺单个上皮细胞悬液制备
Single cell RNA sequencing is a very powerful means for cellular heterogeneous studies and so becoming wildly utilized nowadays. To guarantee the success of such analysis, it is very important, though sometimes difficult, to obtain single cells suspension with high quality, especially from the primary solid organs like mammary glands. Digestion of mouse or human mammary glands with enzymes was previously described. However, the yield, viability, especially the separation degree of the cells have rarely been noticed in these studies. Here we described a detailed protocol for the single epithelial cells suspension preparation from mouse mammary glands, which could be applied for single cell RNA sequencing on different platforms. This protocol could be well adapted for dissociation of other solid organs and tumors, and the single cell suspension could be also used for many other experiments.
ChIP-Seq from Limited Starting Material of K562 Cells and Drosophila Neuroblasts Using Tagmentation Assisted Fragmentation Approach
用Tagmentation Assisted Fragmentation法从K562细胞和果蝇神经母细胞的有限起始材料中提取芯片序列
Chromatin immunoprecipitation is extensively used to investigate the epigenetic profile and transcription factor binding sites in the genome. However, when the starting material is limited, the conventional ChIP-Seq approach cannot be implemented. This protocol describes a method that can be used to generate the chromatin profiles from as low as 100 human or 1,000 Drosophila cells. The method employs tagmentation to fragment the chromatin with concomitant addition of sequencing adaptors. The method generates datasets with high signal to noise ratio and can be subjected to standard tools for ChIP-Seq analysis.
发育生物学
RNA Sequencing of Single Myofibers from Mus musculus
小鼠单肌纤维的RNA序列分析
Whole transcriptome analysis is a key method in biology that allows researchers to determine the effect a condition has on gene regulation. One difficulty in RNA sequencing of muscle is that traditional methods are performed on the whole muscle, but this captures non-myogenic cells that are part of the muscle. In order to analyze only the transcriptome of myofibers we combine single myofiber isolation with SMART-Seq to provide high resolution genome wide expression of a single myofiber.
免疫学
Skin Transplantation and Lymphoid Organ Analysis in Mice
小鼠中皮肤移植和淋巴器官分析
Skin transplantation in mice is an important procedure to evaluate immune responses generated against heterologous grafts, especially given its highly immunogenic nature. In fact, skin is one of the most challenging organs in terms of allograft retention. In this protocol, we provide a detailed procedure for skin grafting using the tail skin as donor organ that is grafted on the dorsal site of thoracic cage in a recipient mouse. We also provide protocols for the systematic analysis of lymphoid organ analysis in transplanted mice. Together these protocols may be valuable for evaluation of parameters that affect skin grafting, including genetic factors, immune cell activation as well as the analysis of compounds that may be useful in allowing graft tolerance.
分子生物学
Assessing Self-interaction of Mammalian Nuclear Proteins by Co-immunoprecipitation
共免疫沉淀法评价哺乳动物核蛋白的自身相互作用
Protein-protein interactions constitute the molecular foundations of virtually all biological processes. Co-immunoprecipitation (CoIP) experiments are probably the most widely used method to probe both heterotypic and homotypic protein-protein interactions. Recent advances in super-resolution microscopy have revealed that several nuclear proteins such as transcription factors are spatially distributed into local high-concentration clusters in mammalian cells, suggesting that many nuclear proteins self-interact. These observations have further underscored the need for orthogonal biochemical approaches for testing if self-association occurs, and if so, what the mechanisms are. Here, we describe a CoIP protocol specifically optimized to test self-association of endogenously tagged nuclear proteins (self-CoIP), and to evaluate the role of nucleic acids in such self-interaction. This protocol has proven reliable and robust in our hands, and it can be used to test both homotypic and heterotypic (CoIP) protein-protein interactions.
Surface Plasmon Resonance Analysis of the Protein-protein Binding Specificity Using Autolab ESPIRIT
利用Autolab ESPIRIT进行表面等离子体共振分析蛋白间结合特异性
Direct protein-protein interactions are known to regulate a wide range of cellular activities. To understand these contacts one can employ various experimental methods like Dynamic Light Scattering (DLS), Fluorescence Resonance Energy Transfer (FRET), Isothermal titration calorimetry (ITC), Chemical crosslinking, Co-immunoprecipitation (Co-IP), Surface Plasmon Resonance (SPR) and many more. Among these, SPR stands out as a quick, label-free, reliable, and accurate quantitation technique. We have used SPR to elucidate the linkage between 14-3-3 Protein 3 (EhP3) and the actin cytoskeleton in the protist pathogen Entamoeba histolytica. It allowed us to screen EhP3 binding with several actin-binding/actin regulatory proteins (Coactosin, Actophorin, Twinfilin, Profilin, and Filamin). Our screening results suggested Coactosin as an important interacting partner of EhP3. A complete kinetic analysis indeed confirmed that EhCoactosin binds EhP3 with an affinity constant of 3 μM.
Super-resolution Microscopy-based Bimolecular Fluorescence Complementation to Study Protein Complex Assembly and Co-localization
基于高分辨率显微镜的双分子荧光互补用于蛋白复合体装配和共定位
Numerous experimental approaches exist to study interactions between two subunits of a large macromolecular complex. However, most methods do not provide spatial and temporal information about binding, which are critical for dissecting the mechanism of assembly of nanosized complexes in vivo. While recent advances in super-resolution microscopy techniques have provided insights into biological structures beyond the diffraction limit, most require extensive expertise and/or special sample preparation, and it is a challenge to extend beyond binary, two color experiments. Using HyVolution, a super-resolution technique that combines confocal microscopy at sub-airy unit pinhole sizes with computational deconvolution, we achieved 140 nm resolution in both live and fixed samples with three colors, including two fluorescent proteins (mTurquoise2 and GFP) with significant spectral overlap that were distinguished by means of shifting the excitation wavelength away from common wavelengths. By combining HyVolution super-resolution fluorescence microscopy with bimolecular fluorescence complementation (SRM-BiFC), we describe a new assay capable of visualizing protein-protein interactions in vivo at sub-diffraction resolution. This method was used to improve our understanding of the ordered assembly of the Saccharomyces cerevisiae spindle pole body (SPB), a ~1 giga-Dalton heteromeric protein complex formed from 18 structural components present in multiple copies. We propose that SRM-BiFC is a powerful tool for examination of direct interactions between protein complex subunits at sub-diffraction resolution in live cells.
神经科学
Induction of Temporal Lobe Epilepsy in Mice with Pilocarpine
毛果芸香碱诱导小鼠颞叶癫痫
In the pilocarpine model of temporal lobe epilepsy (TLE) in rodents, systemic injections of pilocarpine induce continuous, prolonged limbic seizures, a condition termed “Status Epilepticus” (SE). With appropriate doses, many inbred strains of mice show behavioral seizures within an hour after pilocarpine is injected. With the behavioral scoring system based on a modification of the original Racine scale, one can monitor the seizures behaviorally, as they develop into more prolonged seizures and SE. SE is typically associated with damage to subsets of hippocampal neurons and other structural changes in the hippocampus and generally subsides on its own. However, more precise control of the duration of SE is commonly achieved by injecting a benzodiazepine into the mouse 1 to 3 h after the onset of SE to suppress the seizures. Several days following pilocarpine-induced SE, electrographic and behavioral seizures begin to occur spontaneously. The goal of this protocol is to reliably generate mice that develop spontaneous recurrent seizures (SRS) and show the typical neuropathological changes in the brain characteristic of severe human mesial temporal lobe epilepsy (mTLE), without high mortality. To reduce mortality, multiple subthreshold injections of pilocarpine are administered, which increases the percentage of mice developing SE without concomitant mortality. Precise control of the duration of SE (1 or 3 h) is achieved by suppressing SE with the benzodiazepine Midazolam (Versed). We have found that this protocol is an efficient means for generating mice that subsequently develop characteristics of human mTLE including high-frequency interictal spike and wave activity and SRS. In addition, we and others have shown that this protocol produces mice that show excitotoxic cell death of subsets of hippocampal GABAergic interneurons, particularly in the dentate gyrus and compensatory sprouting of excitatory projections from dentate granule cells (mossy fiber sprouting). Aspects of this protocol have been described in several of our previous publications.
Method for Primary Epithelial Cell Culture from the Rat Choroid Plexus
大鼠脉络丛原代上皮细胞培养方法
The choroid plexus consists of a network of secretory epithelial cells localized throughout the lateral, third and fourth ventricles of the brain. Cerebrospinal fluid (CSF) is generated by the choroid plexus and released into the ventricular environment. This biofluid contains an enriched source of proteins, ions, and other signaling molecules for extracellular support of neurons and glial cells within the central nervous system. Given that other cells in the brain also release factors into the CSF, in vitro investigations of choroid plexus function are necessary to isolate processes selectively occurring within and released from this tissue. Here, we describe a protocol to isolate choroid plexus tissue from each of the ventricular locations, and the cell culture conditions required to support growth and maintenance of these epithelial cells. This technique allows for investigations of the functional significance of the choroid plexus, such as for the examination of stimuli promoting the release of growth factors and extracellular vesicles (e.g., exosomes and microvesicles) from ventricle-specific choroid plexus epithelial cells.
Hippocampal Unicellular Recordings and Hippocampal-dependent Innate Behaviors in an Adolescent Mouse Model of Alzheimer’s disease
阿尔茨海默症幼鼠模型中海马单细胞记录和海马依赖先天性行为
Transgenic mice have been used to make valuable contributions to the field of neuroscience and model neurological diseases. The simultaneous functional analysis of hippocampal cell activity combined with hippocampal dependent innate task evaluations provides a reliable experimental approach to detect fine changes during early phases of neurodegeneration. To this aim, we used a merge of patch-clamp with two hippocampal innate behavior tasks. With this experimental approach, whole-cell recordings of CA1 pyramidal cells, combined with hippocampal-dependent innate behaviors, have been crucial for evaluating the early mechanism of neurodegeneration and its consequences. Here, we present our protocol for ex vivo whole-cell recordings of CA1 pyramidal cells and hippocampal dependent innate behaviors in an adolescent (p30) mice.
植物科学
Construction of Antisense RNA-mediated Gene Knock-down Strains in the Cyanobacterium Anabaena sp. PCC 7120
反义RNA介导的鱼腥藻PCC 7120基因敲降株的构建
Anabaena sp. PCC 7120 (hereafter Anabaena) is a model cyanobacterium to study nitrogen fixation, cellular differentiation and several other key biological functions that are analogous in plants. As with any other organism, many genes in Anabaena encode an essential life function and hence cannot be deleted, causing a bottleneck in the elucidation of its genomic function. Antisense RNA (asRNA) mediated approach renders the study of essential genes possible by suppressing (but not completely eliminating) expression of the target gene, thus allowing them to function to some extent. Recently, we have successfully implemented this approach using the strong endogenous promoter of the psbA1 gene (D1 subunit of Photosystem II) introduced into a high-copy replicative plasmid (pAM1956) to suppress the transcript level of the target gene alr0277 (encoding a sigma factor, SigJ/Alr0277) in Anabaena. This protocol represents an efficient and easy procedure to further explore the functional genomics, expanding the scope of basic and applied research in these ecologically important cyanobacteria.
干细胞
Mesenchymal Stromal Cells Derived from Bone Marrow and Adipose Tissue: Isolation, Culture, Characterization and Differentiation
骨髓和脂肪间充质干细胞：分离、培养、鉴定和分化
Since their discovery, mesenchymal stromal cells (MSCs) have received a lot of attention, mainly due to their self-renewal potential and multilineage differentiation capacity. For these reasons, MSCs are a useful tool in cell biology and regenerative medicine. In this article, we describe protocols to isolate MSCs from bone marrow (BM-MSCs) and adipose tissues (AT-MSCs), and methods to culture, characterize, and differentiate MSCs into osteoblasts, adipocytes, and chondrocytes. After the harvesting of cells from bone marrow by flushing the femoral diaphysis and enzymatic digestion of abdominal and inguinal adipose tissues, MSCs are selected by their adherence to the plastic tissue culture dish. Within 7 days, MSCs reach 70% confluence and are ready to be used in subsequent experiments. The protocols described here are easy to perform, cost-efficient, require minimal time, and yield a cell population rich in MSCs.