我々は、科学的な議論や洞察力の提案のために博士キャロリンの価格を感謝したいと思います。この作品は、NIHの助成金AI066095（：畢博リチウムPI）によってサポートされています。

<ol>
	<li>Munoz-Jordan, J. L., Cross, G. A., de Lange, T., Griffith, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=t-loops+at+trypanosome+telomeres.">t-loops at trypanosome telomeres.</a> EMBO J. 20, 579-588 (2001).</li><li>Li, B., Espinal, A., Cross, G. A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypanosome+telomeres+are+protected+by+a+homologue+of+mammalian+TRF2.">Trypanosome telomeres are protected by a homologue of mammalian TRF2.</a> Mol Cell Biol. 25, 5011-5021 (2005).</li><li>Wellinger, R. J., Wolf, A. J., Zakian, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saccharomyces+telomeres+acquire+single-strand+TG1-3+tails+late+in+S+phase.">Saccharomyces telomeres acquire single-strand TG1-3 tails late in S phase.</a> Cell. 72, 51-60 (1993).</li><li>McElligott, R., Wellinger, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+terminal+DNA+structure+of+mammalian+chromosomes.">The terminal DNA structure of mammalian chromosomes.</a> EMBO J. 16, 3705-3714 (1997).</li><li>Harrington, L. A., Greider, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomerase+primer+specificity+and+chromosome+healing.">Telomerase primer specificity and chromosome healing.</a> Nature. 353, 451-454 (1991).</li><li>Jacob, N. K., Skopp, R., Price, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G-overhang+dynamics+at+Tetrahymena+telomeres.">G-overhang dynamics at Tetrahymena telomeres.</a> Embo J. 20, 4299-4308 (2001).</li><li>Barry, J. D., McCulloch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigenic+variation+in+trypanosomes:+enhanced+phenotypic+variation+in+a+eukaryotic+parasite.">Antigenic variation in trypanosomes: enhanced phenotypic variation in a eukaryotic parasite.</a> Adv Parasitol. 49, 1-70 (2001).</li><li>Yang, X., Figueiredo, L. M., Espinal, A., Okubo, E., Li, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RAP1+is+essential+for+silencing+telomeric+variant+surface+glycoprotein+genes+in+Trypanosoma+brucei.">RAP1 is essential for silencing telomeric variant surface glycoprotein genes in Trypanosoma brucei.</a> Cell. 137, 99-109 (2009).</li></ol>

利害の衝突は宣言されません。

トリパノソーマはヒトの睡眠病を引き起こす。この病気は、放置すれば、必然的に致命的である。T.は哺乳動物宿主のトリパノソーマの細胞は、定期的に宿主の免疫攻撃7を回避するために抗原変異を起こす。それゆえにT.を排除することは非常に困難です感染かつてトリパノソーマの細胞が確立されます。我々は最近、テロメアがTの発現の調節に重要な役割を果たすことが実証されているトリパノソーマの表面抗原遺伝子8。したがって、さらにテロメアとT.におけるテロメア維持するためのメカニズムの機能を理解しておくことが大切です。 トリパノソーマの細胞。 テロメアG -オーバーハング構造はテロメラーゼを介したテロメア維持5が不可欠です。テロメアオーバーハングの長さを測定する方法は、テロメアの維持のメカニズムを研究するために有用です。我々は、正常にTを分析するための6】ハイブリダイゼーション3、ライゲーション媒介プライマー伸長ゲルでネイティブを採用しているトリパノソーマテロメアG -オーバーハング構造と塩基テロメア端末の決定のためのアダプターライゲーションの方法。ゲル内の両方のネイティブハイブリダイゼーションとアダプターライゲーションの方法は、細胞内のGのオーバーハングの総量を明らかにすることができますが、唯一のライゲーション媒介プライマー伸長は、正確なGのオーバーハングの長さを明らかにすることができます。 ゲルのハイブリダイゼーションにおけるネイティブの場合は、それは準備ステップを通してのDNAサンプルを変性させることが重要です。これは、TELGオリゴプローブを用いてDNAをハイブリダイズさせることにより制御することができます。 C -オーバーハングはテロメアがないので、ネイティブTELGハイブリダイゼーションは、試料が部分的に変性する限り、任意の信号を得てはならない。テロメアG -オーバーハングは、任意のヌクレアーゼ活性に非常に敏感なのでサンプルはまた、二週間以内にご使用ください。 アダプターライゲーションおよびプライマー伸長のために、精製されたoligoesは明確な結果をもたらす。エキソTはエキソI 3&quot;特定の一本鎖突出のDNAを切断するよりも効果的です。アニールしたサンプルの等しいローディングは、テロメアの総量G -オーバーハングの定量化のために不可欠です。

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<td>SeaKem LE Agarose</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>50004</td>
<td>&nbsp;</td>
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<td>Agarose Type VII (low melting agarose)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A4018</td>
<td>&nbsp;</td>
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<td>Proteinase K, recombinant, PCR grade</td>
<td>&nbsp;</td>
<td>Roche Diagnostic</td>
<td>03 115801001</td>
<td>&nbsp;</td>
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<td>Exo I</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>M0293</td>
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<td>Exo T</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>M0265</td>
<td>&nbsp;</td>
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<td>T7 exonuclease</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>M0263</td>
<td>&nbsp;</td>
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<td>T4 DNA polymerase</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>M0203</td>
<td>&nbsp;</td>
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<td>QIAquick nucleotide removal kit</td>
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examination of the telomere g-overhang structure in trypanosoma brucei

テロメアG -オーバーハング構造は酵母、脊椎動物、およびトリパノソーマを含む多くの真核生物で同定されている。それは 、de novoテロメアDNA合成のためのテロメラーゼの基質として機能し、テロメアの維持のために重要である。T.トリパノソーマは牛で、人間とナガナ病における睡眠病を引き起こす原虫寄生虫です。一度感染哺乳動物宿主、T.トリパノソーマの細胞は、定期的に宿主の免疫攻撃を回避するために、その表面抗原を切り替えます。我々は最近、T.ことが実証されているトリパノソーマテロメアの構造は、T.ために重要な表面抗原の遺伝子発現の調節に重要な役割を果たしているトリパノソーマの病因。しかし、T.トリパノソーマテロメア構造が広範囲にこの特殊な構造の解析のための方法の制限のために研究されていない。我々は正常にネイティブゲル内ハイブリダイゼーションとテロメアG -オーバーハング構造とT.の塩基テロメア端末の決定のためのアダプターのライゲーション法の検討のためのライゲーションを介したプライマー伸長法を採用しているトリパノソーマの細胞。ここで、我々は詳細にプロトコルを説明し、それぞれ異なる長所と限界を比較します。

Watch this Scientific Journal Video about Examination of the Telomere G-overhang Structure in Trypanosoma brucei at JoVE.com

ブルーセイトリパノソーマにおけるテロメア G オーバーハング構造の検査 |免疫学と感染症 |ビデオ

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

テロメアは染色体の安定化に不可欠であり、テロメアG -オーバーハング構造はテロメラーゼを介したテロメア維持に不可欠です。我々は最近ではテロメアG -オーバーハング構造を検出するための2つの方法を採用している<em&gt;トリパノソーマ</emハイブリダイゼーションと説明するライゲーション媒介プライマー伸長、 - ゲルのネイティブである&gt;、。

のテロメアG -オーバーハング構造の検討トリパノソーマ</em

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate ...

examination-of-the-telomere-g-overhang-structure-in-trypanosoma-brucei

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Examination of the Telomere G-overhang Structure in Trypanosoma brucei

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14.2K ビュー.  Cleveland State University. テロメアは染色体の安定化に不可欠であり、テロメアG -オーバーハング構造はテロメラーゼを介したテロメア維持に不可欠です。我々は最近ではテロメアG -オーバーハング構造を検出するための2つの方法を採用しているトリパノソーマ、。

ビデオ: Examination of the Telomere G-overhang Structure in Trypanosoma brucei

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

マイクロ波は、透過型電子顕微鏡による植物ウイルス病の迅速診断をアシスト

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

免疫・感染学

Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States alone, an average of 41,400 deaths and 1.86 million hospitalizations are caused by influenza virus infection each year 1. Point mutations in the polymerase basic protein 2 subunit (PB2) have been linked to the adaptation of the viral infection in humans 2. Findings from such studies have revealed the biological significance of PB2 as a virulence factor, thus highlighting its potential as an antiviral drug target.
The structural genomics program put forth by the National Institute of Allergy and Infectious Disease (NIAID) provides funding to Emerald Bio and three other Pacific Northwest institutions that together make up the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The SSGCID is dedicated to providing the scientific community with three-dimensional protein structures of NIAID category A-C pathogens. Making such structural information available to the scientific community serves to accelerate structure-based drug design.
Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for new lead discovery by targeting a pathway or an entire protein family. Emerald Bio has developed a high-throughput, multi-target parallel processing pipeline (MTPP) for gene-to-structure determination to support the consortium. Here we describe the protocols used to determine the structure of the PB2 subunit from four different influenza A strains.

Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for lead discovery. The following article highlights how the Seattle Structural Genomics Center for Infectious Disease utilizes a multi-target approach for gene-to-structure determination of the PB2 influenza A subunit.

インフルエンザポリメラーゼPB2サブユニットの遺伝子の構造決定のためのマルチターゲット並列処理アプローチ

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

フローサイトメトリーによる胸腺正と負の選択の検討

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

新生児と大人の中枢神経系から培養ミクログリア

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

単離、特徴付けおよび歯肉免疫細胞ネットワークの機能検討

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

High-throughput Gene Tagging in Trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

ハイスループット遺伝子タギングで<em&gt;トリパノソーマ</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

の検出<em&gt;トリパノソーマ</em磁気活性化細胞選別し、フローサイトメトリーによって&gt;バリアント表面糖タンパク質の切り替え

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

The commonality of antibiotic usage in medicine means that understanding the resulting consequences to the host is vital. Antibiotics often decrease host microbiome community diversity and alter the microbial community composition. Many diseases such as antibiotic-associated enterocolitis, inflammatory bowel disease, and metabolic disorders have been linked to a disrupted microbiota. The complex interplay between host, microbiome, and antibiotics needs a tractable model for studying host-microbiome interactions. Our freshwater vertebrate fish serves as a useful model for investigating the universal aspects of mucosal microbiome structure and function as well as analyzing consequential host effects from altering the microbial community. Methods include host challenges such as infection by a known fish pathogen, exposure to fecal or soil microbes, osmotic stress, nitrate toxicity, growth analysis, and measurement of gut motility. These techniques demonstrate a flexible and useful model system for rapid determination of host phenotypes.

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

This study involves methods to reveal effects on a model fish host following alteration of the skin and gut microbiome communities&#160;composition by an antibiotic.

ホスト表現型の検討で<em&gt;カダヤシアフィニス</em&gt;続き抗生物質治療

The commonality of antibiotic usage in medicine means that understanding the resulting consequences to the host is vital. Antibiotics often decrease host ...

Detection of Extravascular Trypanosoma Parasites by Fine Needle Aspiration

Fine Needle Aspiration (FNA) is a routine diagnostic procedure essential to both medical and veterinary practices. It consists of the percutaneous aspiration of cells and/or microorganisms from palpable masses, organs or effusions (fluid accumulation in a body cavity) using a thin needle similar to the regular needle used for the venous puncture. The material collected by FNA is in general highly cellular, and the retrieved aspirate is then smeared, air dried, wet-fixed, stained and observed under a microscope. In the clinical context, FNA is an important diagnostic tool that serves as a guide to the appropriate therapeutic management. Because it is simple, fast, minimally invasive and requires limited investment in the laboratory and human resources, it is extensively used by veterinary practitioners, mostly in domestic, but also in farm animals. In studies using animal models, FNA has the advantage that it can be performed repeatedly in the same animal, enabling longitudinal studies through the collection of cells from tumors and organs/tissues over the course of the disease. In addition to routine microscopy, retrieved material can also be used for immunocytochemistry, electron microscopy, biochemical analysis, flow cytometry, molecular biology or in vitro assays. FNA has been used to identify the protozoan parasite Trypanosoma brucei in the gonads of infected mice, opening the possibility for a future diagnosis in cattle.

Detection of Extravascular Trypanosoma Parasites by Fine Needle Aspiration

Fine Needle Aspiration is a technique, whereby cells are obtained from a lesion or organ using a thin needle. Aspirated material is smeared, stained and examined under a microscope for diagnosis or used for molecular biology, cytometry or in vitro analysis. It is cheap, simple, quick and causes minimal trauma.

細針吸引による血管外トリパノノーマ寄生虫の検出

Fine Needle Aspiration (FNA) is a routine diagnostic procedure essential to both medical and veterinary practices. It consists of the percutaneous ...

Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to proteins of interest. Identification of gene functions in cell line models is important for gene discovery and exploration of cell-intrinsic mechanisms. However, genetic manipulations of immune cells such as T cells and macrophage cell lines using CRISPR/Cas9-mediated knock-in are difficult because of the low transfection efficiency of these cells, especially in a quiescent state. To modify genes in immune cells, drug-resistance selection and viral vectors are typically used to enrich for cells expressing the CRIPSR/Cas9 system, which inevitably results in undesirable intervention of the cells. In a previous study, we designed dual fluorescent reporters coupled to CRISPR/Cas9 that were transiently expressed after electroporation. This technical solution leads to rapid gene deletion in immune cells; however, gene knock-in in immune cells such as T cells and macrophages without the use of drug-resistance selection or viral vectors is even more challenging. In this article, we show that by using cell sorting to aid selection of cells transiently expressing CRISPR/Cas9 constructs targeting the Rosa26 locus in combination with a donor plasmid, gene knock-in can be achieved in both T cells and macrophages without drug-resistance enrichment. As an example, we show how to express human ACE2, a receptor of SARS-Cov-2, which is responsible for the current Covid-19 pandemic, in RAW264.7 macrophages by performing knock-in experiments. Such gene knock-in cells can be widely used for mechanistic studies.

This protocol uses fluorescent reporters and cell sorting to simplify knock-in experiments in macrophage and T cell lines. Two plasmids are used for these simplified knock-in experiments, namely a CRISPR/Cas9- and DsRed2-expressing plasmid and a homologous recombination donor plasmid expressing EBFP2, which is permanently integrated at the Rosa26 locus in immune cells.

CRISPR/Cas9による遺伝子ノックインとマクロファージおよびT細胞株における細胞分類

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to ...