المؤلفان بالشكر دان باس من سيغما الدريخ لعرض الفيديو. ونحن نشكر أيضا دون فرع شيرني Evgenii ، Chesla سكوت ، Hadac اليزابيث ، هاريسون أماندا ، داميان هيثكوت ، أنيكا هولت ، لان تشينغ تشوان ، ماكينتوش سوزانا ، Komarraju Sarvani ، Korchagina ايلينا ، اوليفر كارولين مارتن اولسون ، ستيفن باركر ، Rodinov ايجور الكسندر Tuzikov ويليامز اليانور لنتائجها والمساهمة في التوليف ، وتصميم البنى FSL وتحديد النشاط البيولوجي.

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescein+and+radiolabeled+Function-Spacer-Lipid+constructs+allow+for+simple+in+vitro+and+in+vivo+bioimaging+of+enveloped+virions.">Fluorescein and radiolabeled Function-Spacer-Lipid constructs allow for simple in vitro and in vivo bioimaging of enveloped virions.</a> J Virol Meth. 10, (2011).</li><li>Blake, D., Lan, A., Love, D., Bovin, N., Henry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorophore-kodecytes+-+fluorescent+function-spacer-lipid+(FSL)+modified+cells+for+in+vitro+and+in+vivo+analyses.">Fluorophore-kodecytes - fluorescent function-spacer-lipid (FSL) modified cells for in vitro and in vivo analyses.</a> FEBS J.. 277, 199-199 (2010).</li><li>Harrison, A. L., Olsson, M. L., Brad Jones, R., Ramkumar, S., Sakac, D., Binnington, B., Henry, S., Lingwood, C. A., Branch, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+synthetic+globotriaosylceramide+analogue+inhibits+HIV-1+infection+in+vitro+by+two+mechanisms.">A synthetic globotriaosylceramide analogue inhibits HIV-1 infection in vitro by two mechanisms.</a> Glycobiology. 27, 515-524 (2010).</li><li>Barr, K., Diegel, O., Parker, S., Bovin, N., Henry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function-Spacer-Lipid+(FSL)+constructs+enable+inkjet+printing+of+blood+group+antigens.">Function-Spacer-Lipid (FSL) constructs enable inkjet printing of blood group antigens.</a> FEBS J. 277, 235-235 (2010).</li><li>Lan, C. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Modeling inflammatory bowel disease [dissertation]. , (1988).</li></ol>

ديبورا ألف فارغ وستيفن م. هنري والموظفين وحملة الاسهم من بيوتك المحدودة كودي مالك البراءة التكنولوجيا كودي. دان باس هو موظف من سيغما الدريخ ، والتي رعت الإنتاج من هذه المادة عن طريق الفيديو.

تعديل وخلايا virions مع ثوابت FSL هي تقنية بسيطة جدا وقوية 11/03. لوصف وتمييز FSL بناء خلايا معدلة وغير معدلة virions من الخلايا ، التي تسمى kodecytes وkodevirions 60-10 على التوالي ، ولكن فقط عندما يمكن أن تظهر في مجموعة وظيفية وعرض لتكون موجودة على الغشاء بهم. عندما تتم kodecytes مع تركيزات مختلفة من يبني FSL ، يمكن الإشارة إليها من قبل تركيز المحلول بناء FSL تستخدم لخلق لهم ، على سبيل المثال 15 ميكروغرام / مل وkodecytes ، أو إذا تم استخدام أكثر من واحد لمدة FSL ثم مجتمعة ، مثال : + 7،8 kodecytes البيوتين. عندما خلايا مختلفة وينبغي مقارنة ، بما في ذلك نوع من الخلايا في الوصف ويوصى ، على سبيل المثال البيوتين الخلية الحمراء أو kodecytes kodecytes البيوتين 7،8 بطانة الرحم. وعلى الرغم من أن عملية الإدراج قوية جدا والإدراج (وإن كان ذلك بمعدلات مختلفة) تحدث على نطاق واسع درجة حرارة 4-37 درجة مئوية ، وخلال دقائق إلى ساعات ، للحصول على استنساخه الإدراج التحكم الصارم للوقت الاتصال ، ودرجة الحرارة ، وتركيز FSL ( مطلوبة بما في ذلك صياغة مخفف) وإلى حد أقل تركيز الخلية. حتى الآن ، ويمكن استخدام كافة بنيات FSL لتعديل الخلايا عن طريق نفس الأسلوب 11/04 ، ومع ذلك فمن المتوقع أن ظروف معينة ستكون اكثر ملائمة لتدفق العمل و / أو متطلبات التعامل الأمثل للخلايا / virions إلى تعديل . تركيز FSL الدقيق لتحقيق التأثير المطلوب يحتاج إلى يحدده المستخدم 4. وبناء الخلايا الناتجة FSL تعديل / virions يمكن عادة أن تستخدم بالطريقة نفسها باعتبارها الخلية معدلة / الفيريون في النظم البيولوجية أو تحليلية 4-11. كما سوف يبني FSL أزل ببطء من السطوح عند تعديل على اتصال مع حلول الدهون 7،8 ، أو يتم امتصاصها من قبل خلايا نشطة 11 ، لا بد من النظر في هذه المسائل فيما يتعلق بمستوى النشاط إشارة أو الحصول عليها على مدى فترات من الزمن. يمكن ان تكون ثابتة Kodecytes (على سبيل المثال غلوتارالدهيد / الفورمالين) بعد الإدراج قدمت تثبيتي ، لا تحتوي على دهون ويبلغ حجمه المذيبات وجماعة وظيفية متوافق مع مثبت. يمكن تعديل الخلايا ثابتة بدلا من يبني مع FSL. ويمكن بالإضافة إلى تعديل أسطح الخلية والفيريون أيضا أن يبني FSL غرست مباشرة إلى الدورة الدموية للحيوانات المختبر مما تسبب في تعديل الخلايا الحية المتداولة 7 و 12 لكبح الفيروسات ، والسموم ، و 12 و 7 الأضداد. يبني FLS قد استخدمت أيضا لتزيين الليبوزومات ويمكن طباعتها على أسطح الورق 13 ، حيث شل ، ويمكن استخدامها في فحوصات التشخيص. وينبغي أن القدرة على تعديل الخلايا الحية بسهولة مع مجموعة متزايدة من يبني FSL يثبت أن تكون البحوث مفيدة وأداة للتنمية لدراسة البيولوجيا سطح الخلية.

<img src="/files/ftp_upload/3289/3289table1.jpg" alt="الجدول 1" /> الجدول 1. المتاحة تجاريا يبني FSL وصفها في هذه الورقة.

fsl constructs: a simple method for modifying cell/virion surfaces with a range of biological markers without affecting their viability

القدرة على تعديل / تصور السطوح البيولوجية ، ومن ثم دراسة الخلية المحورة / الفيريون في طائفة من في المختبر والمجراة في بيئات ضروري لكسب مزيد من التبصر في وظيفة معينة أو جزيئات الكيان برمته. وتقتصر عموما دراسات بيولوجية لتعديل السطح الهندسة الوراثية للكائن أو مرفق من الأنصاف التساهمية الكيميائية لسطح الخلية 1،2. لكن هذه التقنيات التقليدية لكشف الخلية المتفاعلات الكيميائية ، أو أنها تتطلب معالجة كبيرة لتحقيق النتائج المرجوة ، مما يجعلها مرهقة ، وأنها قد تؤثر أيضا على البقاء دون قصد / ظائف الخلية المحورة. مطلوب طريقة بسيطة لتعديل دون ان تسبب أذى على سطح الخلايا. في الآونة الأخيرة تقنية جديدة ، فقد طرحت كودي تكنولوجيا مجموعة من يبني رواية تتألف من ثلاثة عناصر : رئيس مجموعة وظيفية (F) ، هل A (S) وذيل المادة الدهنية (L) ويعرف وظيفة ، أو الدهن ، فاصل FSL constructs3. هل تم تحديد (S) لتوفير بناء هذا هو التشتت في الماء ، ولكن سيكون عفويا وستابلي تدرج في غشاء. FSL بناء الأنصاف وظيفية (F) حتى الآن وتشمل مجموعة واسعة من ساتشاريديس الدم بما في ذلك المحددات المتعلقة المجموعة ، والأحماض السياليك ، السكريات hyaluronan ، fluorophores ، البيوتين ، radiolabels ، ومجموعة من الببتيدات 12/03. وقد استخدمت يبني FSL تعديل في الأجنة ، الحيوانات المنوية ، الزرد ، والخلايا الظهارية / بطانة الرحم ، وخلايا الدم الحمراء ، وvirions لإنشاء نظم مراقبة الجودة والألواح التشخيص ، لتعديل خلية التصاق / تفاعل / الانفصال / الشلل ، وعليها في المختبر وفي التصوير من الخلايا الحية / virions 12/03. عملية تعديل الخلايا / virions هي عامة وبسيطة للغاية. الإجراء الأكثر شيوعا هو احتضان خلايا (في وسائل الإعلام الحرة الدهن) حلا لFSL يبني لمدة 1-2 ساعات في 37 درجة مئوية 40-10. أثناء الحضانة وFSL يبني تلقائيا تدرج في الغشاء ، واكتمال العملية. غسل اختيارية. وتعرف الخلايا المعدلة ويبني FSL kodecytes 6-9 ، في حين يتم virions kodevirions 10. FSL يبني كما استخدمت الحقن المباشر وkodecytes / kodevirions في النماذج الحيوانية التجريبية 7،8،10. جميع kodecytes / kodevirions يبدو أن تحتفظ حيويتها الطبيعية وظيفة في حين كسب وظيفة جديدة من طراز F 7،8،10،11 شاردة. مزيج من تشتت حيويا في وسائل الإعلام ، إلى إدماج عفوية أغشية الخلايا ، وسمية منخفضة واضح ، يجعل FSL يبني أدوات بحثية قيمة لدراسة الخلايا وvirions.

Watch this Scientific Journal Video about FSL Constructs: A Simple Method for Modifying Cell/Virion Surfaces with a Range of Biological Markers Without Affecting their Viability at JoVE.com

FSL Constructs: A Simple Method for Modifying Cell/Virion Surfaces with a Range of Biological Markers Without Affecting their Viability

دالة فاصل الدهنية (FSL) يبني يسمح تعديل خصائص سطح الخلايا الحية وvirions دون فقدان للحيوية. الأسلوب يتطلب اتصال بسيط فقط من التوصل إلى حل بناء FSL مع خلية / الفيريون وعفوية ومستقرة التأسيس سطح يحدث.

FSL يبني : طريقة بسيطة لتعديل خلية / الفيريون السطوح مع مجموعة من العلامات البيولوجية دون أن يؤثر ذلك صلاحيتها

The ability to modify/visualize biological surfaces, and then study the modified cell/virion in a range of in vitro and in vivo environments is essential ...

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Research

JoVE Journal

Biology

13.8K Views.  AUT University and KODE Biotech Ltd. دالة فاصل الدهنية (FSL) يبني يسمح تعديل خصائص سطح الخلايا الحية وvirions دون فقدان للحيوية. الأسلوب يتطلب اتصال بسيط فقط من التوصل إلى حل بناء FSL مع خلية / الفيريون وعفوية ومستقرة التأسيس سطح يحدث.

Video: FSL Constructs: A Simple Method for Modifying Cell/Virion Surfaces with a Range of Biological Markers Without Affecting their Viability

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyrosequencing : طريقة بسيطة لالتنميط الجيني دقيقة

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

تطبيق الفحص جيم ايليجانس الدوبامين العصبية للتحلل من التحقق من صحة جينات مرض باركنسون المحتملة

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

Fabrication of Myogenic Engineered Tissue Constructs

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child's small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols[2, 3]. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel&trade;, and NaHCO3. The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size[1, 4, 5]. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures[6]. Once the tissue has solidified at 37&deg;C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1].

تلفيق عضلي يبني الأنسجة المهندسة

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancer-associated glycan variants be exploited for biomarker development aimed at diagnosing early-stage disease. Accordingly, we developed a mass spectrometry-based workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS-14; Agilent). Depleted plasma was trypsin-digested and separated into flow-through and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove N-linked glycans, and analyzed by LC-MS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controls&mdash;fucosylated and sialylated human lactoferrin glycopeptides&mdash;and negative controls&mdash;high mannose glycopeptides from Saccharomyces cerevisiae&mdash;that were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseF-treated glycopeptides from their deamidated Asn-Xxx-Ser/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseF-treatment to ensure full deglycosylation. Future directions include using this workflow to perform mass spectrometry-based discovery experiments on plasma from breast cancer patients and control individuals.

Lectin-conjugated POROS beads were employed for HPLC. Glycopeptide standards served as positive and negative controls. MARS-14 depleted, trypsin-digested human plasma was chromatographed and flow-through (FT) and bound fractions collected for ESI-LC-MS/MS analyses. Glycopeptides were enriched in the bound fraction as compared to FT.

وهناك طريقة HPLC Lectin لإثراء هضميدات انتقائية الغليكوزيلاتي من العينات البيولوجية المعقدة

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the ...

Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood or interstitial fluid); 2) the object is illuminated with light of appropriate wavelength in order to excite the probe into its triplet state; 3) the emitted phosphorescence is collected, and its time course is analyzed to yield the phosphorescence lifetime, which is converted into the oxygen concentration (or partial pressure, pO2). The probe must not interact with the biological environment and in some cases to be 4) excreted from the medium upon the measurement completion. Each of these steps imposes requirements on the molecular design of the phosphorescent probes, which constitute the only invasive component of the measurement protocol. Here we review the design of dendritic phosphorescent nanosensors for oxygen measurements in biological systems. The probes consist of Pt or Pd porphyrin-based polyarylglycine (AG) dendrimers, modified peripherally with polyethylene glycol (PEG's) residues. For effective two-photon excitation, termini of the dendrimers may be modified with two-photon antenna chromophores, which capture the excitation energy and channel it to the triplet cores of the probes via intramolecular FRET (F&ouml;rster Resonance Energy Transfer). We describe the key photophysical properties of the probes and present detailed calibration protocols.

We present principles of oxygen measurements by phosphorescence quenching and review design of porphyrin-based dendritic nanosensors for oxygen imaging in biological systems.

التوليف ومعايرة Nanoprobes الفسفورية للتصوير الأوكسجين في النظم البيولوجية

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

الطفرات والتحليل الوظيفي للقنوات ايون أعربت Heterologously في خلايا الثدييات

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

الوسم نسب خلايا اسماك الزرد مع فلوريسئين الليزر ديكستران Uncagable

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4, 5. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6, 7, the mosquitoes Aedes aegypti and Anopheles gambiae 8, 9 and the tobacco hornworm, Manduca sexta 10, 11. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12.
Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13.

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

A بروتوكول بسيط لاستخراج اليرقات من Hemocytes البرية

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

Recombineering مثلي التركيبات التوحد في<em&gt; ذبابة الفاكهة</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

A Method for Microinjection of Patiria minata Zygotes

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and evolution of developmental processes. The sea star, Patiria miniata, is gaining prevalence as a model system for these types of studies which were previously performed almost exclusively in the sea urchins, Strongylocentrotus purpuratus and Lytechinus variegatus. An advantage of these model systems is the ease of producing modified embryos in which a particular gene is up or downregulated, labeling a group of cells, or introducing a reporter gene. A single microinjection method is capable of creating a wide variety of such modified embryos. Here, we present a method for obtaining gametes from P. miniata, producing zygotes, and introducing perturbing reagents via microinjection. Healthy morphant embryos are subsequently isolated for quantitative and qualitative studies of gene function. The availability of genome and transcriptome data for this organism has increased the types of studies that are performed and the ease of executing them.

A Method for Microinjection of Patiria minata Zygotes

Methods that produce morphant embryos are essential to study developmental mechanisms and gene regulatory networks. The sea star Patiria miniata is an emerging model system for these studies. Here we present a protocol for obtaining gametes, producing cultures of embryos, and rapid microinjection of zygotes from this species.

منهج في Microinjection من<em&gt; Patiria minata</em&gt; بيضات ملقحة

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and ...

FSL يبني : طريقة بسيطة لتعديل خلية / الفيريون السطوح مع مجموعة من العلامات البيولوجية دون أن يؤثر ذلك صلاحيتها

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