केन्द्रीय तंत्रिका तंत्र से Transducing कक्ष के लिए lentiviral वैक्टर का उत्पादन
Summary
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> इन विट्रो में</em> और<em> Vivo में</em>.
Abstract
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 (Ψ) 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 (http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf). 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 (http://www.addgene.org/). 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.
Protocol
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Name of the reagent | Company | Catalogue number |
| DMEM | Sigma-Aldrich | D5796 |
| MEM | Invitrogen | 11090-081 |
| Fetal bovine serum | Hyclone | SV3001403 |
| PBS | Mediatech | 21-040-CM |
| Trypsin-EDTA | Sigma-Aldrich | T3924 |
| Sodium butyrate | Sigma-Aldrich | B5887 |
| Hexadimethrine bromide (Polybrene) | Sigma-Aldrich | H9268 |
| 293T cells | ATCC | CRL-11268 |
| HT1080 cells | ATCC | CCL-121 |
| Falcon 100 x 20 mm tissue culture dish | BD Biosciences | 353003 |
| 1 x 3 ½ in polyallomoer centrifuge tube | Beckman-Coulter | 326823 |
| 0.2-micron syringe filter | Corning | 431219 |
| QIAamp DNA Mini Kit | Qiagen | 51304 |
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