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

وأيد هذا العمل من قبل المعاهد الوطنية للصحة منح العلوم العصبية الأساسية مخطط (P30 NS057105، المدارس الأردنية البريطانية) لجامعة واشنطن، برنامج مشروع المنح NS032636 (المدارس الأردنية البريطانية)، ومركز الأمل للاضطرابات العصبية.

1. التعبئة والتغليف من ناقلات Lentiviral ويتم إنتاج النواقل Lentiviral بواسطة cotransfection من ناقلات نقل lentiviral والبلازميدات الأخرى اللازمة للتغليف في الخلايا 293T بواسطة فوسفات الكالسيوم طريقة ترنسفكأيشن. نحن نستخدم 10 زراعة الأنسجة 100 ملم أطباق ف...

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في هذا البروتوكول، لقد أظهرنا إنتاج النواقل lentiviral وتطبيق هذه النواقل في الثقافات القشرة المخية الحديثة. أثبتنا كفاءة وخلية نوع محدد تنبيغ مع الناقلة التي تنتجها هذه الأساليب. عندما يتم استخدام المروج synapsin، GFP التعبير بدقة الخلايا العصبية المحددة. عندما يتم استخدام ?...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> فهرس العدد </td></tr><tr><td> DMEM </td><td> سيغما الدريخ </td><td> D5796 </td></tr><tr><td> MEM </td><td> إينفيتروجن </td><td> 11090-081 </td></tr><tr><td> مصل بقري جنيني </td><td> Hyclone </td><td> SV3001403 </td></tr><tr><td> برنامج تلفزيوني </td><td> Mediatech </td><td> 21-040-CM </td></tr><tr><td> التربسين-EDTA </td><td> سيغما الدريخ </td><td> T3924 </td></tr><tr><td> الصوديوم الزبدات </td><td> سيغما الدريخ </td><td> B5887 </td></tr><tr><td> Hexadimethrine بروميد (Polybrene) </td><td> سيغما الدريخ </td><td> H9268 </td></tr><tr><td> 293T الخلايا </td><td> ATCC </td><td> CRL-11268 </td></tr><tr><td> HT1080 الخلايا </td><td> ATCC </td><td> CCL-121 </td></tr><tr><td> فالكون 100 × 20 ملم أنسجة الثقافةه طبق </td><td> العلوم البيولوجية دينار بحريني </td><td> 353003 </td></tr><tr><td> 1 × 3 ½ في أنبوب الطرد المركزي polyallomoer </td><td> بيكمان كولتر، </td><td> 326823 </td></tr><tr><td> 0.2 ميكرون حقنة فلتر </td><td> كورنينج </td><td> 431219 </td></tr><tr><td> QIAamp DNA البسيطة كيت </td><td> QIAGEN </td><td> 51304 </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

في هذا البروتوكول وصفنا الإنتاج، وتنقية ومعايرة ناقلات lentiviral. ونحن نقدم مثالا على lentiviral إيصال الجينات ناقلات بوساطة في الخلايا العصبية مثقف الابتدائي والنجمية. وقد طرقنا تنطبق أيضا على أنواع الخلايا الأخرى<em&gt; في المختبر</em&gt; و<em&gt; في الجسم الحي</em&gt;.

انتاج ناقلات Lentiviral للخلايا Transducing من الجهاز العصبي المركزي

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

Research

JoVE Journal

Neuroscience

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

النظامية والمحلية تسليم المخدرات لعلاج أمراض الجهاز العصبي المركزي في المجموعات القوارض

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution ...

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

التصوير المتعدد الوسائط من زرع الخلايا الجذعية في الجهاز العصبي المركزي من الفئران

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

وضع العلامات واحدة من الخلايا في الجهاز العصبي المركزي<em&gt; ذبابة الفاكهة الدروسوفيلا</em

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

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

مباشرة داخل البطين تسليم المخدرات الى القوارض الجهاز العصبي المركزي

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

تحليل المناعى في الجهاز العصبي المركزي والمحيطي الجرذ العقدة الليمفاوية أقسام الأنسجة

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

في الجسم الحي Optogenetic تحفيز الجهاز العصبي المركزي القوارض

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

المقاصة البصري للنظام العصبي المركزي الماوس عن طريق وضوح السلبي

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

تسجيل الكهربية لنشاط الجهاز العصبي المركزي للطور الثالث Melanogaster المورفولوجية

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

زرع الذرية القشرية الهندسية كيميائياً في عقول الفئران المبكرة بعد الولادة

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

توصيل البراز المضاد للندرة في الجهاز العصبي المركزي للفئران

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