Name: production of lentiviral vectors for transducing cells from the central nervous system
Uploaded: 2012-05-24T13:00:00
Description: Watch this Scientific Journal Video about Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System at JoVE.com

इस काम में NIH न्यूरोसाइंस खाका कोर वाशिंगटन विश्वविद्यालय, प्रोग्राम परियोजना अनुदान NS032636 (BJS) के लिए अनुदान (NS057105 p30, BJS) के द्वारा और मस्तिष्क संबंधी विकार के लिए आशा है कि केंद्र द्वारा समर्थित किया गया.

1. Lentiviral वैक्टर की पैकेजिंग Lentiviral वैक्टर हैं एक lentiviral हस्तांतरण के वेक्टर और अन्य को 293T कोशिकाओं में कैल्शियम फॉस्फेट अभिकर्मक विधि द्वारा पैकेजिंग के लिए आवश्यक प्लास्मिड का cotransfection से उत्पादित कर ...

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इस प्रोटोकॉल में, हम neocortical संस्कृतियों में इन वैक्टर lentiviral वैक्टर और आवेदन का उत्पादन दिखाया गया है. हम इन तरीकों द्वारा उत्पादित वैक्टर के साथ कुशल और सेल transduction टाइप विशिष्ट प्रदर्शन किया. जब synapsin प्रमोटर प...

<table border="1"><tbody><tr><td> अभिकर्मक का नाम </td><td> कंपनी </td><td> सूचीपत्र संख्या </td></tr><tr><td> DMEM </td><td> सिग्मा Aldrich </td><td> D5796 </td></tr><tr><td> सदस्य </td><td> Invitrogen </td><td> 11090-081 </td></tr><tr><td> भ्रूण गोजातीय सीरम </td><td> Hyclone </td><td> SV3001403 </td></tr><tr><td> पीबीएस </td><td> Mediatech </td><td> 21-040 - मुख्यमंत्री </td></tr><tr><td> Trypsin - EDTA </td><td> सिग्मा Aldrich </td><td> T3924 </td></tr><tr><td> सोडियम butyrate </td><td> सिग्मा Aldrich </td><td> B5887 </td></tr><tr><td> Hexadimethrine ब्रोमाइड (Polybrene) </td><td> सिग्मा Aldrich </td><td> H9268 </td></tr><tr><td> 293T कोशिकाओं </td><td> ATCC </td><td> CRL - 11,268 </td></tr><tr><td> HT1080 कोशिकाओं </td><td> ATCC </td><td> सीसीएल-121 </td></tr><tr><td> फाल्कन 100 x 20 मिमी ऊतक संस्कृतियोंई पकवान </td><td> बी.डी. बायोसाइंसेज </td><td> 353,003 </td></tr><tr><td> 1 एक्स 3 साढ़े polyallomoer अपकेंद्रित्र ट्यूब में </td><td> के Beckman-कल्टर </td><td> 326,823 </td></tr><tr><td> 0.2 माइक्रोन सिरिंज फिल्टर </td><td> कोर्निंग </td><td> 431,219 </td></tr><tr><td> QIAamp डीएनए मिनी किट </td><td> क्विएज़न </td><td> 51,304 </td></tr></tbody></table>

production of lentiviral vectors for transducing cells from the central nervous system

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

Watch this Scientific Journal Video about Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System at JoVE.com

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

इस प्रोटोकॉल में हम उत्पादन, शोधन, और lentiviral वैक्टर का अनुमापन का वर्णन. हम lentiviral वेक्टर की मध्यस्थता प्राथमिक सभ्य न्यूरॉन्स और astrocytes में जीन वितरण का एक उदाहरण प्रदान करते हैं. हमारे तरीकों में भी अन्य प्रकार की कोशिकाओं को लागू कर सकते हैं<em&gt; इन विट्रो में</em&gt; और<em&gt; Vivo में</em&gt;.

केन्द्रीय तंत्रिका तंत्र से Transducing कक्ष के लिए lentiviral वैक्टर का उत्पादन

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Research

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Neuroscience

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

प्रणालीगत और स्थानीय कृंतक मॉडल में केन्द्रीय तंत्रिका तंत्र के रोगों के इलाज के लिए दवा वितरण

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During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

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In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

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कृंतक केन्द्रीय तंत्रिका तंत्र के लिए ड्रग्स का प्रत्यक्ष intraventricular डिलिवरी

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

चूहा केंद्रीय तंत्रिका तंत्र में Immunohistochemical विश्लेषण और परिधीय लिम्फ नोड ऊतक वर्गों

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

कृंतक केंद्रीय तंत्रिका तंत्र के विवो optogenetic उत्तेजना में

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

माउस केंद्रीय तंत्रिका तंत्र के ऑप्टिकल क्लियरिंग निष्क्रिय स्पष्टता का उपयोग

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar  Drosophila Melanogaster

तीसरी-Instar Drosophila Melanogaster के केंद्रीय तंत्रिका तंत्र गतिविधि की Electrophysiological रिकॉर्डिंग

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

प्रारंभिक प्रसवोत्तर माउस दिमाग में रसायनजन्य रूप से इंजीनियर कॉर्टिकल इंटरन्यूरॉन संततिकाका का प्रत्यारोपण

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System

चूहा केंद्रीय तंत्रिका तंत्र में एंटीसेंस ओलिगोन्यूक्लिओटाइड्स का अंत:केंद्र वितरण

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central ...