慢病毒包装教程Lentivirus Packaging and Production.docx

1、慢病毒包装教程Lentivirus Packaging and ProductionLentivirus Packaging and Production The laboratories of Didier Trono (EPFL) and Robert Weinberg (Whitehead Institute) have deposited plasmids for the production of lentiviral particles. These plasmids can be used with many lentiviral vectors, including The R

2、NAi Consortium shRNA vectors being distributed by Sigma (i.e. MISSION shRNAs) and Open Biosystems (i.e. TRC shRNAs). Overview For producing lentiviral particles, you typically need three components: 1) a lentiviral vector, such as pLKO.1 or pLVTHM, containing the shRNA or transgene, 2) a packaging v

3、ector, such as psPAX2 or pCMV-dR8.2 dvpr, and 3) an envelope vector, such as pMD2.G or pCMV-VSVG. For most applications, you can produce viral particles by transient transfection of 293T cells with a 2nd generation packaging system (e.g. packaging plasmid psPAX2 and envelope plasmid pMD2.G). 2nd Gen

4、eration Packaging System In general, lentiviral vectors with a wildtype 5 LTR need the 2nd generation packaging system because these vectors require TAT for activation. All lentiviral vectors from the Trono or Aebischer lab require packaging with a 2nd generation system. Below are two 2nd generation

5、 systems. Lentiviral plasmids based on pLKO.1 can be packaged with either system, although the first system has been reported to produce higher titer. See Addgenes pLKO.1 Protocol for producing lentiviral particles. 2nd generation system deposited by the Trono lab: ID/FONT PlasmidDescription12260 ps

6、PAX22nd generation packaging plasmid for producing viral particles. psPAX2 contains a robust CAG promoter for efficient expression of packaging proteins. Trono lab and Aebischer lab lentiviral vectors require psPAX2. Produces higher titer than pCMV-dR8.2 dvpr. 12259 pMD2.GEnvelope plasmid for produc

7、ing viral particles 2nd generation system deposited by the Weinberg lab: ID/FONT PlasmidDescription8455 pCMV-dR8.2 dvpr2nd generation packaging plasmid for producing viral particles 8454 pCMV-VSVGEnvelope plasmid for producing viral particles 3rd Generation Packaging System The 3rd generation packag

8、ing system offers maximal biosafety but is more cumbersome to use, as it involves the transfection of four different plasmids in the producer cells (two packaging plasmids, an envelope plasmid, and the lentiviral vector). If you wish to use this system, you need to have a lentiviral vector with a ch

9、imeric 5 LTR in which the HIV promoter is replaced with CMV or RSV, thus making it TAT-independent. Examples of these vectors include pLKO.1, pLL3.7, pLB, pLenti6, pSico, pCL, and pCS. Most Aebischer and Trono Lab lentiviral vectors CANNOT be used with this system. A lentiviral vector carrying a chi

10、meric 5 LTR can be packaged with either the 2nd or 3rd generation packaging system. ID/FONT PlasmidDescription12251 pMDLg/pRRE3rd generation packaging plasmid for producing viral particles 12253 pRSV-Rev3rd generation packaging plasmid for producing viral particles 12259 pMD2.GEnvelope plasmid for p

11、roducing viral particles More information Click here to browse other RNAi vectors, or search for plasmids using the search bar at the top of the page. Trono Lab website or Lentiweb: information and a discussion forum on cloning, packaging, and other protocols. Moffat J et. al. 2006. A lentiviral RNA

12、i library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124:1283-1298. (PubMed) Ventura et. al. 2004. Cre-lox-regulated conditional RNA interference from transgenes. PNAS 2004 Jul 13;101(28):10380-5. (PubMed) Naldini L et. al. 1996. In vivo gene delivery and stable

13、transduction of nondividing cells by a lentiviral vector. Science 272:263-267. (PubMed) Dull et al., A Third-Generation Lentivirus Vector with a Conditional Packaging System. J. Virol. 1998 72(11): 8463-8472. (PubMed) Zufferey R et. al. 1997. Multiply attenuated lentiviral vector achieves efficient

14、gene delivery in vivo. Nat Biotechnol 15(9):871-5. (PubMed) Zufferey R et. al. 1998. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol 72(12):9873-80. (PubMed) Cell Line The 293T cell line for producing lentiviral particles can be obtained from GenHunter. pLKO

15、.1 Protocol pLKO.1 - TRC Cloning Vector Addgene Plasmid 10878. Protocol Version 1.0. December 2006. Copyright Addgene 2006, All Rights Reserved. This protocol is provided for your convenience. See warranty information in appendix. Click here for a printable copy. Table of Contents A. pLKO.1-TRC Cloning Vector o A.1 The RNAi Consortium o A.2 Map of pLKO.1 o A.3 Related plasmids B. Designing shRNA Oligos for pLKO.1 o B.1 Determine the optimal 21-mer targets in your gene o B.2 Order oligos compatible with pLKO.1 C. Cloning shRNA oligos into pLKO.1 o C.1 Recommended materials o C.2 Annealing