Name: transient expression of proteins by hydrodynamic gene delivery in mice
Uploaded: 2014-05-05T16:30:00
Description: Watch this Scientific Journal Video about Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice at JoVE.com

हम शुरू में हमें प्रौद्योगिकी के विभिन्न पहलुओं के बारे में अपने निरंतर मार्गदर्शन के लिए HGD तकनीक और डॉ. रसेल थॉमसन (मेडिसिन के अल्बर्ट आइंस्टीन कॉलेज) को पढ़ाने के लिए ज़िया लियू (रीजनरोन फार्मास्यूटिकल्स) धन्यवाद. इस काम के हंटर कॉलेज द्वारा वित्त पोषित किया गया, CUNY धनराशि शुरू और NSF रोटी पुरस्कार IOS-1249166. हम माउस यकृत पर ऊतक विज्ञान के लिए NYULMC हिस्तोपैथोलोजी कोर NYUCI केंद्र सहायता अनुदान, एनआईएच / NCI 5 P30CA16087-31 धन्यवाद.

यहाँ वर्णित सभी प्रयोगों हंटर कॉलेज के संस्थागत पशु की देखभाल और उपयोग समिति, न्यूयॉर्क के सिटी विश्वविद्यालय द्वारा अनुमोदित किया गया. Endotoxin मुक्त प्लास्मिड डीएनए के 1. तैयारी <ol><li> एक हौसले स...

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B., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+impact+of+hydrodynamic+injection+on+mouse+liver.">Structural impact of hydrodynamic injection on mouse liver.</a> Gene Ther. 14, 129-137 (2007).</li></ol>

सही ढंग से प्रदर्शन करते हैं, तो HGD transgene प्रसव के एक उल्लेखनीय सुरक्षित और कारगर साधन है. सफल HGD के लिए महत्वपूर्ण कदम हैं: 1) कम से कम 8 सेकंड में माउस 3) की पूंछ नस में) खारा वाहन 2 की एक बड़ी मात्रा में डीएनए की स?...

द्वारा अपनी पहली विवरण के बाद लियू एट अल. और झांग एट अल., Hydrodynamic जीन डिलीवरी (HGD) कृंतक मॉडल प्रणाली 1, 2 में जीन समारोह के अध्ययन के लिए एक अमूल्य उपकरण बन गया है. तकनीक लक्ष्य अंगों 1, 2 की कोशिकाओं से डीएनए प्लाज्मिड के तेज की सुविधा के लिए चूहों की पूंछ नस में समाधान की एक बड़ी मात्रा (8-12% शरीर के वजन) के तेजी से इंजेक्शन (5-7 सेकंड) शामिल है. इन शर्तों गुर्दा, तिल्ली, फेफड़े और दिल में जिगर और कम जीन अभिव्यक्ति में मजबूत जीन की अभिव्यक्ति के लिए नेतृत्व. 10 माइक्रोग्राम pCMV-lacZ प्लाज्मिड इंजेक्शन HGD तारीख 1 सबसे कुशल, गैर वायरल, vivo जीन वितरण पद्धति है, जिससे hepatocytes के रूप में ज्यादा के रूप में 40% transfect कर सकते हैं. वायरल वाहक के विपरीत, pDNA तैयार करने के लिए आसान है, कृंतक मेजबान 3 में एक प्रतिरक्षा प्रतिक्रिया प्रकाश में लाना नहीं करता है और अंत के साथ पुनर्संयोजन द्वारा एक स्वास्थ्य खतरा उत्पन्न नहीं करताogenous वायरस. HGD ने दिया डीएनए अणु पैकेजिंग की जरूरत नहीं है, क्योंकि इसके अलावा, इस विधि 150 केबी के रूप में 4 के रूप में बड़े जीवाणु कृत्रिम गुणसूत्रों (बीएसी) की डिलीवरी के लिए उपयुक्त है. एक hydrodynamic विधि द्वारा दिया गया है कि अणुओं के अन्य प्रकार शाही सेना 5-10, morpholinos 11, प्रोटीन 12, 13 और अन्य छोटे अणुओं 12, 14 में शामिल हैं. अन्य प्रसव के तरीके से अधिक HGD के फायदे और नुकसान साहित्य 15-20 में उत्कृष्ट समीक्षा में चर्चा की गई है और लेखकों में से एक नंबर प्रक्रिया 21-23 का विस्तृत विवरण उपलब्ध कराई है. HGD द्वारा चूहों में transgenes परिचय सुरक्षित और 1-3, 24 से प्रभावी है और विधि तुलनीय सफलता 25 के साथ चूहों में इस्तेमाल किया गया है. कुछ संशोधनों के साथ, सबूत की अवधारणा प्रयोगों मुर्गियों 26, रब में बाहर किया गया हैबड़े जानवरों में इस तकनीक के vivo आवेदन एक चुनौती बनी हुई है, हालांकि, 27 और सूअरों 28 बिट्स. इस पद्धति का उपयोग करते हैं, तो एक और सामान्य सीमा उपलब्ध स्तनधारी अभिव्यक्ति वैक्टर के कई जीन अभिव्यक्ति का एक लगातार उच्च स्तर को प्राप्त करने के लिए घटकों की कमी है. लक्ष्य अंगों में एक pCMV ल्यूक प्लाज्मिड, जीन अभिव्यक्ति का उपयोग हालांकि, प्रारंभिक, उच्च अभिव्यक्ति के स्तर इंजेक्शन 1 के बाद पहले सप्ताह में तेजी से चला जाता है, HGD के बाद दस मिनट के रूप में जल्दी के रूप में स्पष्ट है. लंबे समय तक transgene अभिव्यक्ति प्लाज्मिड डिजाइन 3, 24 बहरहाल, उच्च स्तर के जीन की अभिव्यक्ति के रखरखाव अक्सर दोहराया आवश्यकता इंजेक्शन में इस्तेमाल प्रमोटर और Intron के आधार पर संभव है. इस कारण से, HGD हानिकारक प्रोटीन या प्रोटीन उत्पादों के लिए लंबी अवधि के निवेश का एक परिणाम हैं कि पुराने रोगों का अध्ययन करने के लिए कम उपयुक्त हो सकता है. इन सीमाओं के साथ, HGD पीओ के अध्ययन के लिए एक असाधारण शक्तिशाली उपकरण हैएक जीन और यह के प्रभाव की क्षमता का भूमिका (समीक्षा के लिए, 15 देखें) विवो, साथ ही चिकित्सीय प्रभाव और प्रोटीन के नियमन में और रोग के पशु मॉडल की स्थापना के लिए म्यूटेंट. उदाहरण के लिए, HGD व्यक्तिगत रूप से संबंधित जीन बाहर दस्तक दी है कि चूहों में विभिन्न जीन निर्माणों को शुरू करने से डोमेन और प्रोटीन के अमीनो एसिड के लिए समारोह आवंटित करने के लिए इस्तेमाल किया जा सकता है. इसके अलावा, इस तकनीक को किसी भी माउस तनाव में इस्तेमाल किया जा सकता है. वितरण की नस, इंजेक्शन की मात्रा और गति में सही सुई प्रविष्टि: इस प्रोटोकॉल सफल अभिकर्मक प्राप्त करने के लिए आवश्यक तकनीकी पहलुओं पर ध्यान देने के साथ चूहों में HGD वर्णन करता है. इस विधि के आवेदन अफ्रीकी trypanosomiasis, मनुष्यों की एक घातक रोग और पशुधन 29, 30 के एक माउस मॉडल में प्रदर्शन किया है. Trypanosomes की कई प्रजातियों के पशुओं में रोग का कारण हैं, सबसे ख में प्रतिरक्षा परिसरों जन्मजात की वजह से मानव में बीमारी का कारण नहीं कर सकतेlood बुलाया असिकाय अपघट्य कारकों (TLFs) 29, 31, 32. ये ताकना के गठन, उच्च घनत्व लिपो प्रोटीन (एचडीएल) दो अद्वितीय, रहनुमा विशेष प्रोटीन होते हैं. Trypanosomes में TLFs के तेज जो सुविधा HPR, ligand,, और Apol-मैं, अपघट्य घटक 31, 33-38 ट्रिपैनोसोमा ब्रुसे rhodesiense के कारण बांध और मानव Apol-मैं 34, 39 neutralizes कि एक सीरम प्रतिरोध जुड़े प्रोटीन (एसआरए) की अभिव्यक्ति के लिए मनुष्यों को संक्रमित करने में सक्षम है. लंगूर TLF कारण इसकी मुक़्तलिफ़ Apol-I प्रोटीन से 40 एसआरए द्वारा निष्प्रभावी नहीं है. एक स्तनधारी अभिव्यक्ति वेक्टर (pRG977), चूहों में लंगूर TLF घटकों के ट्रांसजेनिक अभिव्यक्ति का उपयोग करते हुए, जैसा कि पहले बताया मानव संक्रामक trypanosomes 40 के खिलाफ संरक्षण प्रदान करता है. यहाँ प्रस्तुत प्रतिनिधि डेटा hydrodynamic जीन डिलीवरी एक प्रोटीन की चिकित्सीय प्रभाव का अध्ययन करने के लिए लागू किया जा सकता है कि कैसे प्रदर्शित करता है.

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</table>

transient expression of proteins by hydrodynamic gene delivery in mice

विवो में transgenes की कुशल अभिव्यक्ति जीन समारोह का अध्ययन और रोगों के उपचार को विकसित करने में महत्वपूर्ण महत्व का है. पिछले वर्षों में, hydrodynamic जीन डिलीवरी (HGD) कृन्तकों में transgenes देने के लिए एक सरल, तेज, सुरक्षित और प्रभावी तरीके के रूप में उभरा है. इस तकनीक perfused अंगों की कोशिका झिल्ली की पारगम्यता बढ़ाने के लिए शारीरिक समाधान की एक बड़ी मात्रा का तेजी से इंजेक्शन द्वारा उत्पन्न बल पर निर्भर करता है और इस प्रकार की कोशिकाओं में डीएनए उद्धार. HGD का मुख्य लाभ में से एक नग्न प्लास्मिड डीएनए (pDNA) का उपयोग स्तनधारी कोशिकाओं में transgenes शुरू करने की क्षमता है. एक प्लाज्मिड का उपयोग कर एक exogenous जीन परिचय उल्लेखनीय सुरक्षित, न्यूनतम अत्यधिक कुशल, श्रमसाध्य और वायरल वाहक के विपरीत है. HGD शुरू में चूहों में जीन देने के लिए इस्तेमाल किया गया था, यह अब oligonucleotides, कृत्रिम क्रोमोसोम, शाही सेना, प्रोटीन और चूहों में छोटे अणुओं सहित पदार्थों की एक विस्तृत श्रृंखला, चूहों देने के लिए प्रयोग किया जाता हैऔर, एक सीमित हद तक अन्य जानवरों. इस प्रोटोकॉल चूहों में HGD वर्णन करता है और सफलतापूर्वक प्रक्रिया के प्रदर्शन के लिए महत्वपूर्ण हैं कि विधि के तीन प्रमुख पहलुओं पर केंद्रित है: नस में सुई की सही प्रविष्टि, इंजेक्शन की मात्रा और वितरण की गति. उदाहरण स्रावित, रहनुमा विशेष प्रोटीन सांकेतिक शब्दों में बदलना है कि दो जीन की क्षणिक अभिव्यक्ति के लिए इस विधि के आवेदन को दिखाने के लिए दिया जाता है, अपोलीपोप्रोटीन ली (Apol मैं) और haptoglobin से संबंधित प्रोटीन (HPR).

माउस की पूंछ नस में सुई की सही स्थिति (चित्रा 1 में सचित्र) सफलतापूर्वक हाइड्रोइनेमिकस आधारित अभिकर्मक से एक transgene पहुंचाने के लिए एक शर्त है. इंजेक्शन की पूरी मात्रा एक 6-8 की अवधि के भीतर दिया जा सकत?...

Watch this Scientific Journal Video about Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice at JoVE.com

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Hydrodynamic जीन डिलीवरी द्वारा नग्न डीएनए के vivo अभिकर्मक में कम से कम भड़काऊ प्रतिक्रिया के साथ एक पशु के ऊतकों में जीनों का परिचय. जीन उत्पाद के लिए पर्याप्त मात्रा में जीन समारोह और विनियमन के साथ ही प्रोटीन संरचना और समारोह का विश्लेषण किया जा सकता है कि इस तरह उत्पन्न कर रहे हैं.

चूहे में द्रव्यगतिकी जीन डिलीवरी द्वारा प्रोटीन की क्षणिक अभिव्यक्ति

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

transient-expression-of-proteins-by-hydrodynamic-gene-delivery-in-mice

Research

JoVE Journal

Biology

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

प्राथमिक electroporation के लिए murine अस्थि मज्जा से hematopoietic सेल संस्कृतियों की तैयारी

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

कार्बनिक सॉल्वेंट और एसिटिक एसिड के बिना जी 250 Coomassie के साथ जैल में प्रोटीन की धुंधला

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

जीन Pulser MXcell electroporation प्रणाली का उपयोग करने के लिए उच्च क्षमता के साथ प्राथमिक कोशिकाओं transfect

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

एक स्वचालित सेल काउंटर का उपयोग करने के लिए जीन एक्सप्रेशन अध्ययन सरल: आईएल-4 निर्भर जीन एक्सप्रेशन के Namalwa कक्ष में siRNA नॉकडाउन

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

के आयल - एंबेडेड और जमे हुए वर्गों ड्रोसोफिला वयस्क स्नायु

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

सक्रिय टी. के पुनर्गठन इन विट्रो में castaneum टेलोमिरेज

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

पुनः संयोजक प्रोटीन का उच्च स्तर क्षणिक अभिव्यक्ति के लिए पौधे के कुशल Agroinfiltration

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Hypoxia निम्नलिखित एकल कोशिका के स्तर पर ग्लोबल शाही सेना संश्लेषण का विश्लेषण

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

मेटाबोलिक दर के मापन में<em&gt; ड्रोसोफिला</em&gt; Respirometry का उपयोग

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.