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Çeşitli çalışmalarda intraserebral infüzyon, doku travması ve kan-beyin bariyerini hakaret teslim kanül boyutunu azaltarak (Perry ve ark, 1993) azalır olduğunu göstermiştir, inflamasyonu azalttığı ve bağışıklık tepkisi zayıflatılmış (pisi et al , 1991) ve reaktif gliozis (Nikkhah ve ark, 1994) azalır. Biz burada 6 kat, geleneksel olarak kullanılan teslim kanüller ile karşılaştırıldığında azalır çapında bir mikrokanül ile beynin içinde nöroaktif ajanlar kronik teslimat için bir yöntem mevcut. Bu sistem, zaman ve lokalize travma ve reaktif gliozis düzeyi önemli ölçüde azalır üzerinde güvenilir bir temsilcisi çözüm sağladığını göstermiştir. Bu çalışmaların nihai bir ucu çapı 50 m teslim kanül çalışan, ancak daha küçük uç çapları kullanılan olabilir. Bu tür mikroinfüzyon sistemlerin birincil erdem infüzyon çevresinde minimal travma ödemeden, çok küçük, ayrık hedeflere ajanlar sunmak için yeteneğidir. Açıklayan infüzyon sistemi yapımında kullanılan microcannulas, önceden belirlenen uzunluklarda ve uç çapları kolayca özel yapılmış olabilir. Mikroinfüzyon sistemi, güvenli, düşük profilli fiksasyon sağlayan ve büyük bir kafatası kaide gerek kalmaz hayvanın kafatası yüzeyine uyacak şekilde tasarlanmıştır. Bu nedenle cerrahi yara kolayca böylece hayvan rahatsızlık ve enfeksiyon riskini azaltarak, akıcı bir mikroinfüzyon kaide üzerinde sütüre edilebilir. Buna ek olarak, kafatası sistem güvenliğini daha az kafatası yüzey alanı gerektirir; bu nedenle, ek veya daha sonraki işlemler, daha büyük bir kaide müdahalesi olmaksızın yapılabilir. Burada açıklanan metodoloji istihdam bir adet Ancak, hesap belli teknik değerlendirmeler içine almalıdır. Daha yüksek seviyede beceri ve üretim ve implantasyon prosedürü daha az zaman verimli daha standart infüzyon sistemleri için olabilir. Öte yandan, bu yüksek hassasiyetli tekniği uygulayarak ayrılan zaman (ve hayvanlar), galip ya da en azından dengeli olabilir. Başka bir zorluk mikrokanül hemen hemen her çap ile özelleştirilmiş olabilir, çok küçük bir lümen mikrokanül infusate bileşenleri veya implantasyon (örneğin, kan, beyin dokusu) sırasında karşılaşılan doku tarafından tıkanır olabilir. Bu risk mikrokanül ucu implantasyon prosedürü sırasında (infusate çözünenlerin kurumasını önlemek için su ya da tuzlu doymuş pamuklu çubukla kullanarak, örneğin) nemlendirilmiş tutarak, &quot;kan-free&quot; cerrahi temiz performans azalır. MOP yüklenebilir çözüm özellikle kompozisyon ve / veya viskozite için uygun bir mikrokanül ucu çapı kurmak araştırmacı tavsiye ederiz. Bu kolay bir protokol (Şekil 4) açıklanan benzer vitro test sistemi kullanılarak elde ya da monte edilen sistem, sadece 37 ° C tuzlu su çeker ve banyo bileşimi ve / izleme veya hacmini ölçerek zamanla MOP içeriğinin. Bir dezavantajı ucu çapı çok küçük (örneğin, ~ 10-50 mikron), özellikle mikrokanül nispeten kırılgan olmasıdır. Genellikle ameliyat sırasında yanlışlıkla mikrokanül kırarak, tüm sistemin değiştirilmesi olmasını gerektirir, bu birim tamir veya yeniden inşa olarak cerrahi süresi önemli ölçüde uzanacak. Bu tür teknikler yetenekli bilim adamları ve ancak nadiren uygulama titiz yöntemi deneyim kırılması,. Deney bitiminde, mikroinfüzyon sistemi mikrokanül hasar ve bunun patentini kalmıştır edilmemiş olduğunu sağlamak için kontrol edilebilir. Nöroaktif ajan uygun miktarda teslim edilmiştir sağlamak için mini-osmotik pompa hacmi de ölçülebilir, ancak, geri dönüşüm veya yeniden pompaları tavsiye etmiyoruz. Mevcut yöntemlerden yetişkin sıçanlarda gösterilmiştir iken, travma azaltır ve sabitleme için az kafatası yüzey alanı, fareler ya da genç sıçanlar gibi küçük hayvanlar, deneyler için geleneksel yöntemleri üzerinde tercih olarak kabul edilebilir gerektiren bir sistem kullanın. Şimdiki yöntem biraz daha ileri düzeyde deneyci tarafından yeterlilik ve beceri gerektirmesine rağmen, belki de bir büyüklük sırasına hassasiyetini arttırmak ve ilgi bölgeye travma ile ilişkili deneysel boşa en aza indirmek için sunmaktadır. Laboratuvarımızda, biz bulduk, daha güvenilir ve sağlam etkileri deneysel müdahale doğru çevirir.

A correction was made to: <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/716/">Construction and Implantation of a Microinfusion System for Sustained Delivery of Neuroactive Agents.</a> A key reference was excluded and a revised abstract was republished.
Additional Reference: 
22. Cunningham, M.G., Ames, H.M., Donalds, R.A., &amp; Benes, F.M. Construction and implantation of a microinfusion system for sustained delivery of neuroactive agents. Journal of Neuroscience Methods 167, 213-220, doi:10.1016/j.jneumeth.2007.08.016 (2008).
Revised Abstract:
Sustained delivery of neuroactive agents is widely used in neuroscience, but poses many technical challenges. It is necessary to deliver the agent with high precision while minimizing localized trauma and inflammation. Also, the ability to customize the system to accommodate animals of different species and sizes is desirable. This video presentation demonstrates the construction of an infusion system that can be fitted to any particular research animal. The delivery microcannula diameter is approximately 10-fold smaller than most infusion cannulas presently used. This translates into enhanced accuracy and reduced trauma to the brain region under study. The delivery cannula can also be sculpted to fit the contour of the surface of the animal's skull, thereby allowing closure of the scalp incision neatly over the infusion system, precluding the need for a skull-mounted pedestal, reducing risk of infection, and ensuring a greater level of comfort to the animal. The system is assembled in an air-free environment and requires the researcher to fashion glass micropipettes with a heat source. These construction methods require special skills that are best acquired, if not in person, using video instruction. (This article is based on work first reported in J Neurosci Methods. 2008 Jan 30;167(2):213-20. Epub 2007 Aug 28.).
Original Abstract:
Experimental protocols used for chronic infusion of neuroactive agents within regions of the brain often utilize a mini-osmotic pump system. Agents are commonly delivered via a stainless steel cannula with a diameter of 0.30 mm or greater. Systems utilizing a cannula of this caliber may impose trauma to the area of interest resulting in architectural damage, thereby compromising structural integrity and normal functioning. As neuroscience inquiry becomes more sophisticated, investigation of brain structures and circuitry requires improved levels of accuracy and higher resolution. We have developed a method for the preparation and implantation of a chronic infusion system within the brain utilizing a borosilicate microcannula with a tip diameter of 50 microns. This technique reduces damage to the local environment and diminishes reactive gliosis at the site of infusion. The configuration of the microinfusion system is also able to conform to the surface of the animal's skull, precluding the need for large cranial pedestals, thus facilitating closure of the scalp incision and reducing the risk of infection. We demonstrate reliable sustained delivery of a dye having a representative molecular weight using an in vitro model and in vivo studies in rats.

A correction was made to: <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/716/">Construction and Implantation of a Microinfusion System for Sustained Delivery of Neuroactive Agents.</a> A key reference was excluded and a revised abstract was republished.

Erratum: Construction and Implantation of a Microinfusion System for Sustained Delivery of Neuroactive Agents.

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<td>Mini-osmotic pumps (MOPs)</td>
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<td>Microforceps</td>
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construction and implantation of a microinfusion system for sustained delivery of neuroactive agents.

Nöroaktif ajanlar kronik infüzyon için kullanılan deneysel protokolleri beyin bölgeleri içinde sık sık bir mini-osmotik pompa sistemi kullanmaktadır. Ajanlar, yaygın olarak, 0,30 mm veya daha büyük bir çapa sahip paslanmaz çelik bir kanül aracılığı ile teslim edilir. Bu çapta bir kanül kullanan sistemler, böylece yapısal bütünlüğünü ve normal işleyişi ödün, mimari hasara neden ilgi alanı travma getirebilir. Nörobilim soruşturma hale geldikçe, daha sofistike, beyin yapılarının incelenmesi ve devreleri, doğruluk ve daha yüksek çözünürlük gelişmiş düzeyde gerektirir. Biz bir ucu çapı 50 mikron ile borosilikat mikrokanül kullanarak beynin içinde kronik bir infüzyon sistemi hazırlanması ve implantasyon için bir yöntem geliştirdiler. Bu teknik, yerel çevreye zarar azaltır ve infüzyon yerinde reaktif gliozis azalır. Mikroinfüzyon sistem konfigürasyonu da skalp insizyonu kapatılmasını kolaylaştırmak ve böylece enfeksiyon riskini azaltır, büyük kafatası kaideleri gerek kalmaz, hayvanın kafatası yüzeyine uyacak şekilde yapabiliyor. Biz, in vitro modeli ve in vivo çalışmalar sıçanlarda kullanarak bir temsilci moleküler ağırlığa sahip bir boya güvenilir sürekli teslim göstermektedir.

Watch this Scientific Journal Video about Construction and Implantation of a Microinfusion System for Sustained Delivery of Neuroactive Agents. at JoVE.com

Construction and Implantation of a Microinfusion System for Sustained Delivery of Neuroactive Agents.

Nörobilim soruşturma hale geldikçe, daha sofistike, beyin yapılarının incelenmesi ve devreleri, doğruluk ve daha yüksek çözünürlük gelişmiş düzeyde gerektirir. Biz bir ucu çapı 50 mikron ile borosilikat mikrokanül kullanarak beynin içinde kronik bir infüzyon sistemi hazırlanması ve implantasyon için bir yöntem geliştirdiler.

Nöroaktif Acenteleri Sürekli teslim için bir mikroinfüzyon Sistemi İnşaat ve İmplantasyon.

Sustained delivery of neuroactive agents is widely used in neuroscience, but poses many technical challenges. It is necessary to deliver the agent with ...

construction-implantation-microinfusion-system-for-sustained-delivery

Nanyang Technological University

Research

JoVE Journal

Biology

15.4K Görüntüleme.  Harvard Medical School. Nörobilim soruşturma hale geldikçe, daha sofistike, beyin yapılarının incelenmesi ve devreleri, doğruluk ve daha yüksek çözünürlük gelişmiş düzeyde gerektirir. Biz bir ucu çapı 50 mikron ile borosilikat mikrokanül kullanarak beynin içinde kronik bir infüzyon sistemi hazırlanması ve implantasyon için bir yöntem geliştirdiler.

Video: Construction and Implantation of a Microinfusion System for Sustained Delivery of Neuroactive Agents.

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

Sıçan Kalp Engineered Doku implantasyonu

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Biyoloji

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to elaborate distinct aspects of motor function and to determine even subtle loss of movement capacity. To enhance efficacy and resolution of testing, tests should permit qualitative and quantitative measures of motor function and be sensitive to changes in performance during recovery periods. The present study describes a new task to assess skilled walking in the rat to measure both forelimb and hindlimb function at the same time. Animals are required to walk along a horizontal ladder on which the spacing of the rungs is variable and is periodically changed. Changes in rung spacing prevent animals from learning the absolute and relative location of the rungs and so minimize the ability of the animals to compensate for impairments through learning. In addition, changing the spacing between the rungs allows the test to be used repeatedly in long-term studies. Methods are described for both quantitative and qualitative description of both fore- and hindlimb performance, including limb placing, stepping, co-ordination. Furthermore, use of compensatory strategies is indicated by missteps or compensatory steps in response to another limb&#x2019;s misplacement. 

The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.

Görev Yürüyüş Merdiven Basamak: Puanlama Sistemi ve Pratik Uygulama.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

High Speed Droplet-based Delivery System for Passive Pumping in Microfluidic Devices

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery system. Passive pumping is the phenomenon by which surface tension induced pressure differences drive fluid movement in closed channels. The automated fluid delivery system consists of a set of voltage controlled valves with micro-nozzles connected to a fluid reservoir and a control system. These voltage controlled valves offer a volumetrically precise way to deliver fluid droplets to the inlet of a microfluidic device in a high frequency manner. Based on the  dimensions demonstrated in the current study example, the system is capable of flowing 4 milliliters per minute (through a 2.2mm by 260um cross-sectional channel). Based on these same channel dimensions, fluid exchange of a point inside the channel can be achieved in as little as eight milliseconds. It is observed that there is interplay between momentum of the system (imparted by a combination of the droplets created by the valves and the fluid velocity in the channel), and the surface tension of the liquid. Where momentum provides velocity to the fluid flow (or vice-versa), equilibration of surface tension at the inlet provides a sudden stop to any flow. This sudden stop allows the user to control the flow characteristics of the channel and opens the door for a variety of biological applications, ranging anywhere from reagent delivery to drug-cell studies. It is also observed that when nozzles are aimed at the inlet at shallow angles, the droplet momentum can cause additional interesting fluid phenomena, such as mixing of multiple droplets in the inlet.

A novel microfluidic system has been developed using the phenomenon of passive pumping and a user controlled fluid delivery system. This microfluidic system has the potential to be used in a wide variety of biological applications given its low cost, ease of use, volumetric precision, high speed, repeatability and automation.

Mikroakışkan Cihazlar Pasif pompalanması için Yüksek Hızlı Damlacık tabanlı Dağıtım Sistemi

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

Görselleştirme Caenorhabditis Elegans Tırnak Yapıları Lipofilik Vital Boya, DII

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Için bir sistem<em&gt; Ex vivo</emEmbriyonik Pankreas&gt; ekimi

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

Bilinçli Farelerde Glomerüler Filtrasyon Hızı Ölçümü için bir Yüksek verimli Yöntemi

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

Phosphopeptide Analysis of Rodent Epididymal Spermatozoa

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once the pre-cursor round cell begins to elongate and differentiate into what is morphologically recognized as a spermatozoon. However, the spermatozoon is very immature, having no ability for motility or egg recognition. Both of these events occur once the spermatozoa transit a secondary organ known as the epididymis. During the ~12 day passage that it takes for a sperm cell to pass through the epididymis, post-translational modifications of existing proteins play a pivotal role in the maturation of the cell. One major facet of such is protein phosphorylation. In order to characterize phosphorylation events taking place during sperm maturation, both pure sperm cell populations and pre-fractionation of phosphopeptides must be established. Using back flushing techniques, a method for the isolation of pure spermatozoa of high quality and yield from the distal or caudal epididymides is outlined. The steps for solubilization, digestion, and pre-fractionation of sperm phosphopeptides through TiO2 affinity chromatography are explained. Once isolated, phosphopeptides can be injected into MS to identify both protein phosphorylation events on specific amino acid residues and quantify the levels of phosphorylation taking place during the sperm maturation processes.

Proteomic analysis of any cell type is highly dependent on both purity and pre-fractionation of the starting material in order to de-complexify the sample prior to liquid chromatography mass spectrometry (MS). By using back-flushing techniques, pure spermatozoa can be obtained from rodents. Following digestion, phosphopeptides can be enriched using TiO2.

Kemirgen Epididimal Spermatozoa&#39;lar phosphopeptide Analizi

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once ...

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

Alt klonlama Artı Ekleme (SPI) - Gen Hedefleme Vektörler Rapid İnşaat için Yeni Bir Yöntem Recombineering

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...

Single Cell Collection of Trophoblast Cells in Peri-implantation Stage Human Embryos

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a critical yet enigmatic biological event that has been historically difficult to study due to technical and ethical limitations. Implantation is initiated by the development of the trophectoderm to early trophoblast and subsequent differentiation into distinct trophoblast sublineages. Aberrant early trophoblast differentiation may lead to implantation failure, placental pathologies, fetal abnormalities, and miscarriage. Recently, methods have been developed to allow human embryos to grow until day 13 post-fertilization in vitro in the absence of maternal tissues, a time-period that encompasses the implantation period in humans. This has given researchers the opportunity to investigate human implantation and recapitulate the dynamics of trophoblast differentiation during this critical period without confounding maternal influences and avoiding inherent obstacles to study early embryo differentiation events in vivo. To characterize different trophoblast sublineages during implantation, we have adopted existing two-dimensional (2D) extended culture methods and developed a procedure to enzymatically digest and isolate different types of trophoblast cells for downstream assays. Embryos cultured in 2D conditions have a relatively flattened morphology and may be suboptimal in modeling in vivo three-dimensional (3D) embryonic architectures. However, trophoblast differentiation seems to be less affected as demonstrated by anticipated morphology and gene expression changes over the course of extended culture. Different trophoblast sublineages, including cytotrophoblast, syncytiotrophoblast and migratory trophoblast can be separated by size, location, and temporal emergence, and used for further characterization or experimentation. Investigation of these early trophoblast cells may be instrumental in understanding human implantation, treating common placental pathologies, and mitigating the incidence of pregnancy loss.

Here, we describe a method for warming vitrified human blastocysts, culturing them through the implantation period in vitro, digesting them into single cells and collecting early trophoblast cells for further investigation.

Peri-implantasyon Aşamasında Trophoblast Hücrelerinin Tek Hücre Li Hücre Koleksiyonu İnsan Embriyoları

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a ...

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular compartments. This paper describes an enzyme protection assay to study the extent of S. aureus internalization and its intracellular survival in adherent non-professional phagocytic cells (NPPCs) as well as the intracellular efficacy of antimicrobial compounds. NPPCs are grown in a multi-well plate until they reach 100% confluence. S. aureus cultures are grown overnight in cell culture medium. The bacterial suspension is diluted according to the number of cells per well to inoculate the cells at a controlled multiplicity of infection. Inoculated cells are incubated for 2 h to allow the bacteria to be internalized by the NPPCs, following which lysostaphin is added to the culture medium to selectively kill extracellular bacteria. Lysostaphin is present in the culture medium for the rest of the experiment. 
At this point, the infected cells could be incubated with antimicrobial compounds to assess their intracellular activities against S. aureus. Next, the cells are washed three times to remove the drugs, and intracellular S. aureus load is then quantified by culturing on agar plates. Alternatively, for studying staphylococcal virulence factors involved in intracellular survival and cell toxicity, lysostaphin could be inactivated with proteinase K to eliminate the need for washing steps. This tip improves the reliability of the intracellular bacterial load quantification, especially if cells tend to detach from the culture plate when they become heavily infected because of the multiplication of intracellular S. aureus. These protocols can be used with virtually all types of adherent NPPCs and with 3D cell culture models such as organoids.

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

This protocol aims to describe how to study the extent of Staphylococcus aureus internalization and its ability to survive inside the human host cell, as well as the intracellular efficacy of antimicrobial compounds.

Antimikrobiyal Bileşiklerin Staphylococcus aureus Internalization ve Hücre İçi Etkinliğini İncelemek için Geliştirilmiş Enzim Koruma Tahlilleri