Bio-protocol最新文献

Many protein families consist of multiple highly homologous proteins, whether they are encoded by different genes or originating from the same genomic location. Predominance of certain isoforms has been linked to various pathological conditions, such as cancer. Detection and relative quantification of protein isoforms in research are commonly done via immunoblotting, immunohistochemistry, or immunofluorescence, where antibodies against an isoform-specific epitope of particular family members are used. However, isoform-specific antibodies are not always available, making it impossible to decipher isoform-specific protein expression patterns. Here, we describe the insertion of the versatile 11 amino acid HiBiT tag into the genomic location of the protein of interest. This tag was developed and is distributed by Promega (Fitchburg, WI, USA). This protocol describes precise and specific protein expression analysis of highly homologous proteins through expression of the HiBiT tag, enabling protein expression quantification when specific antibodies are missing. Protein expression can be analyzed through traditional methods such as western blotting or immunofluorescence, and also in a luciferase binary reporter system, allowing for reliable and fast relative expression quantification using a plate reader. Graphical overview.

The immune-inhibitory molecule programmed cell death ligand 1 (PD-L1) has been shown to play a role in pathologies such as autoimmunity, infections, and cancer. The expression of PD-L1 not only on cancer cells but also on non-transformed host cells is known to be associated with cancer progression. Generation of PD-L1 deficiency in the murine system enables us to specifically study the role of PD-L1 in physiological processes and diseases. One of the most versatile and easy to use site-specific gene editing tools is the CRISPR/Cas9 system, which is based on an RNA-guided nuclease system. Similar to its predecessors, the Zinc finger nucleases or transcription activator-like effector nucleases (TALENs), CRISPR/Cas9 catalyzes double-strand DNA breaks, which can result in frameshift mutations due to random nucleotide insertions or deletions via non-homologous end joining (NHEJ). Furthermore, although less frequently, CRISPR/Cas9 can lead to insertion of defined sequences due to homology-directed repair (HDR) in the presence of a suitable template. Here, we describe a protocol for the knockout of PD-L1 in the murine C57BL/6 background using CRISPR/Cas9. Targeting of exon 3 coupled with the insertion of a HindIII restriction site leads to a premature stop codon and a loss-of-function phenotype. We describe the targeting strategy as well as founder screening, genotyping, and phenotyping. In comparison to NHEJ-based strategy, the presented approach results in a defined stop codon with comparable efficiency and timelines as NHEJ, generates convenient founder screening and genotyping options, and can be swiftly adapted to other targets.

An efficient cell culture system for hepatitis B virus (HBV) is indispensable for research on viral characteristics and antiviral agents. Currently, for HBV infection assays in cell culture, HBV genome-integrated cell line-derived viruses are commonly used. However, these viruses are not suitable for the evaluation of polymorphism-dependent viral characteristics or resistant mutations against anti-viral agents. To detect the infection of cell culture-generated HBV (HBVcc) by the transient transfection of the HBV molecular clone, a large amount of purified viruses is needed, because such viruses exhibit limited infection efficiencies in cell culture. Here, we describe how to generate and purify HBVcc by the transient transfection of HBV molecular clones. This system provides a powerful tool for studying the infection and propagation of HBV and for developing anti-viral agents against HBV.

In vivo microscopy of plants with high-frequency imaging allows observation and characterization of the dynamic responses of plants to stimuli. It provides access to responses that could not be observed by imaging at a given time point. Such methods are particularly suitable for the observation of fast cellular events such as membrane potential changes. Classical measurement of membrane potential by probe impaling gives quantitative and precise measurements. However, it is invasive, requires specialized equipment, and only allows measurement of one cell at a time. To circumvent some of these limitations, we developed a method to relatively quantify membrane potential variations in Arabidopsis thaliana roots using the fluorescence of the voltage reporter DISBAC₂(3). In this protocol, we describe how to prepare experiments for agar media and microfluidics, and we detail the image analysis. We take an example of the rapid plasma membrane depolarization induced by the phytohormone auxin to illustrate the method. Relative membrane potential measurements using DISBAC₂(3) fluorescence increase the spatio-temporal resolution of the measurements and are non-invasive and suitable for live imaging of growing roots. Studying membrane potential with a more flexible method allows to efficiently combine mature electrophysiology literature and new molecular knowledge to achieve a better understanding of plant behaviors. Key features Non-invasive method to relatively quantify membrane potential in plant roots. Method suitable for imaging seedlings root in agar or liquid medium. Straightforward quantification.

During the first meiotic prophase in mouse, repair of SPO11-induced DNA double-strand breaks (DSBs), facilitating homologous chromosome synapsis, is essential to successfully complete the first meiotic cell division. Recombinases RAD51 and DMC1 play an important role in homology search, but their mechanistic contribution to this process is not fully understood. Super-resolution, single-molecule imaging of RAD51 and DMC1 provides detailed information on recombinase accumulation on DSBs during meiotic prophase. Here, we present a detailed protocol of recombination foci analysis of three-color direct stochastic optical reconstruction microscopy (dSTORM) imaging of SYCP3, RAD51, and DMC1, fluorescently labeled by antibody staining in mouse spermatocytes. This protocol consists of sample preparation, data acquisition, pre-processing, and data analysis. The sample preparation procedure includes an updated version of the nuclear spreading of mouse testicular cells, followed by immunocytochemistry and the preparation steps for dSTORM imaging. Data acquisition consists of three-color dSTORM imaging, which is extensively described. The pre-processing that converts fluorescent signals to localization data also includes channel alignment and image reconstruction, after which regions of interest (ROIs) are identified based on RAD51 and/or DMC1 localization patterns. The data analysis steps then require processing of the fluorescent signal localization within these ROIs into discrete nanofoci, which can be further analyzed. This multistep approach enables the systematic investigation of spatial distributions of proteins associated with individual DSB sites and can be easily adapted for analyses of other foci-forming proteins. All computational scripts and software are freely accessible, making them available to a broad audience. Key features Preparation of spread nuclei, resulting in a flattened preparation with easy antibody-accessible chromatin-associated proteins on dSTORM-compatible coverslips. dSTORM analysis of immunofluorescent repair foci in meiotic prophase nuclei. Detailed descriptions of data acquisition, (pre-)processing, and nanofoci feature analysis applicable to all proteins that assemble in immunodetection as discrete foci. Graphical overview.

Tension and force propagation play a central role in tissue morphogenesis, as they enable sub- and supra-cellular shape changes required for the generation of new structures. Force is often generated by the cytoskeleton, which forms complex meshworks that reach cell-cell or cell-extracellular matrix junctions to induce cellular rearrangements. These mechanical properties can be measured through laser microdissection, which concentrates energy in the tissue of interest, disrupting its cytoskeleton. If the tissue is undergoing tension, this cut will induce a recoil in the surrounding regions of the cut. This protocol describes how one can perform laser microdissection experiments and subsequently measure the recoil speed of the sample of interest. While we explain how to carry out these experiments in Drosophila embryos, the recoil calibration and downstream analyses can be applied to other types of preparations. Key features Allows measuring tension in live Drosophila embryos with a relatively simple approach. Describes a quick way to mount a high number of embryos. Includes a segmentation-free recoil quantification that reduces bias and speeds up analysis. Graphical overview.

Microtubule structure is commonly investigated using single-particle analysis (SPA) or subtomogram averaging (STA), whose main objectives are to gather high-resolution information on the αβ-tubulin heterodimer and on its interactions with neighboring molecules within the microtubule lattice. The maps derived from SPA approaches usually delineate a continuous organization of the αβ-tubulin heterodimer that alternate regularly head-to-tail along protofilaments, and that share homotypic lateral interactions between monomers (α-α, β-β), except at one unique region called the seam, made of heterotypic ones (α-β, β-α). However, this textbook description of the microtubule lattice has been challenged over the years by several studies that revealed the presence of multi-seams in microtubules assembled in vitro from purified tubulin. To analyze in deeper detail their intrinsic structural heterogeneity, we have developed a segmented subtomogram averaging (SSTA) strategy on microtubules decorated with kinesin motor-domains that bind every αβ-tubulin heterodimer. Individual protofilaments and microtubule centers are modeled, and sub-volumes are extracted at every kinesin motor domain position to obtain full subtomogram averages of the microtubules. The model is divided into shorter segments, and subtomogram averages of each segment are calculated using the main parameters of the full-length microtubule settings as a template. This approach reveals changes in the number and location of seams within individual microtubules assembled in vitro from purified tubulin and in Xenopus egg cytoplasmic extracts. Key features This protocol builds upon the method developed by J.M. Heumann to perform subtomogram averages of microtubules and extends it to divide them into shorter segments. Microtubules are decorated with kinesin motor-domains to determine the underlying organization of its constituent αβ-tubulin heterodimers. The SSTA approach allows analysis of the structural heterogeneity of individual microtubules and reveals multi-seams and changes in their number and location within their shaft. Graphical overview.

Epigenetic modifications of DNA, and especially cytosine, play a crucial role in regulating basic cellular processes and thereby the overall cellular metabolism. Their levels change during organismic and cellular development, but especially also in pathogenic aberrations such as cancer. Levels of respective modifications are often addressed in bulk by specialized mass spectrometry techniques or by employing dedicated ChIP-seq protocols, with the latter giving information about the sequence context of the modification. However, to address modification levels on a single cell basis, high- or low-content microscopy techniques remain the preferred methodology. The protocol presented here describes a straightforward method to detect and quantify different DNA modifications in human cell lines, which can also be adapted to other cultured mammalian cell types. To this end, cells are immunostained against two different cytosine modifications in combination with DNA counterstaining. Image acquisition takes place on a confocal microscopy system. A semi-automated analysis pipeline helps to gather data in a fast and reliable fashion. The protocol is comparatively simple, fast, and cost effective. By employing methodologies that are often well established in most molecular biology laboratories, many researchers are able to apply the described protocol straight away in-house.

Immotile cilia of crown cells at the node of mouse embryos are required for sensing leftward fluid flow that gives rise to the breaking of left-right (L-R) symmetry. The flow-sensing mechanism has long remained elusive, mainly because of difficulties inherent in manipulating and precisely analyzing the cilium. Recent progress in optical microscopy and biophysical analysis has allowed us to study the mechanical signals involving primary cilia. In this study, we used high-resolution imaging with mechanical modeling to assess the membrane tension in a single cilium. Optical tweezers, a technique used to trap sub-micron-sized particles with a highly focused laser beam, allowed us to manipulate individual cilia. Super-resolution microscopy allowed us to discern the precise localization of ciliary proteins. Using this protocol, we provide a method for applying these techniques to cilia in mouse embryonic nodes. This method is widely applicable to the determination of mechanical signals in other cilia.

Synapses provide the main route of signal transduction within neuronal networks. Many factors regulate critical synaptic functions. These include presynaptic calcium channels, triggering neurotransmitter release, and postsynaptic ionotropic receptors, mediating excitatory and inhibitory postsynaptic potentials. The key features of synaptic transmission and plasticity can be studied in primary cultured hippocampal neurons. Here, we describe a protocol for the preparation and electrophysiological analysis of paired hippocampal neurons. This model system allows the selective genetic manipulation of one neuron in a simple neuronal network formed by only two hippocampal neurons. Bi-directionally analyzing synaptic transmission and short-term synaptic plasticity allows the analysis of both pre- and postsynaptic effects on synaptic transmission. For example, with one single paired network synaptic responses induced by both, a wild-type neuron and a genetically modified neuron can be directly compared. Ultimately, this protocol allows experimental modulation and hence investigation of synaptic mechanisms and thereby improves previously developed methods of studying synaptic transmission and plasticity in ex vivo cultured neurons. Key features Preparation of ex vivo paired cultured hippocampal neurons. Bi-directional electrophysiological recordings of synaptic transmission and plasticity. Genetic modulation of synaptic network formation (demonstrated by presynaptic viral overexpression of the auxiliary calcium channel α₂δ-2 subunit). Graphical overview.