Generation of Stable Expression Mammalian Cell Lines Using Lentivirus
利用慢病毒制备稳定表达哺乳动物细胞系
发布: 2018年11月05日第8卷第21期 DOI: 10.21769/BioProtoc.3073 浏览次数: 22895
评审: Ingrid LinVarun KesherwaniAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
May 2015
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Abstract
Lentiviruses are used very widely to generate stable expression mammalian cell lines. They are used for both gene down-regulation (by using shRNA) or for gene up-regulation (by using ORF of gene of interest). The technique of generating stable cell lines using 3rd generation lentivirus is very robust and it typically takes about 1-2 weeks to get stable expression for most mammalian cell lines. The advantage of using the 3rd generation lentivirus are that are very safe and they are replication incompetent.
Keywords: Lentivirus (慢病毒)
Stable cell lines (稳定细胞系)
Gene knockdown (基因敲低)
Gene over-expression (基因过表达)
Mammalian cell lines (哺乳动物细胞系)
Transduction (转染)
Background
As compared to the short term protein expression resulting from transient transfection, stable cell lines generated using lentivirus exhibit long term protein expression and the system is highly reproducible (LaGory et al., 2015). Below is the outline for generation of stable cell lines using lentivirus (Figure 1). In this protocol, we describe the use of the 3rd generation lentiviral system which uses three different plasmids for generating stable cell lines. First plasmid contains your gene of interest usually flanked by Long Terminal Repeat (LTR) sequences, that are integrated into the host genome. A variety of these plasmids can be obtained from Addgene. Second is the pCMV Delta R8.2 plasmid that encodes all components necessary for packaging the lentivirus viz. HIV-1 Gag, Pol, Tat and Rev. Third and the final plasmid is the pCMV VSVG that encodes the viral envelope protein. 
Figure 1. Schematic representation of generation of stable cell lines using lentivirus
Materials and Reagents
- 10 cm2 tissue culture treated plates (Corning, catalog number: CLS430165)
- Low protein binding 0.45 μm PDVF filters (Millipore, catalog number: SLHV033RS)
- 10 ml plastic syringes sterile (any brand)
- Sterile Glass jar
- Disposable plastic pipettes (5 ml and 10 ml)
- Sterile 1.5 ml Eppendorf tubes
- Pasteur pipettes
- HEK293T (293T) (ATCC, catalog number: CRL-3216)
- Target cell line (any human cancer cell line)
- Xtreme gene 9 transfection reagent (Sigma-Aldrich, 636577900)
- Packaging plasmids: pCMV delta R8.2 (Addgene, catalog number: 12263) and pCMV-VSV-G (Addgene, catalog number: 8454) and your plasmid of interest (test and control plasmid – see Notes)
- 10% bleach
- Polybrene solution: 8 mg/ml in water filter sterilized (Hexadimethrine bromide, Sigma, catalog number: H9268), made fresh every time
- Opti-MEM reduced serum medium (Thermo Fisher, catalog number: 31985062)
- DMEM complete medium with 10% FBS and pen-strep
- Selection antibiotic (e.g., Puromycin)
- Dry ice or liquid nitrogen
- DMSO
- Liquid nitrogen
Equipment
- Cell culture incubators (37 °C, 5% CO2 and 32 °C, 5% CO2)
- Hemocytometer (Standard)
- Laminar hood BSL-2 type
- 37 °C water bath
- Autoclave
- Micropipettes (1,000 μl, 200 μl)
- -80 °C freezer
Procedure
文章信息
版权信息
© 2018 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Tandon, N., Thakkar, K. N., LaGory, E. L., Liu, Y. and Giaccia, A. J. (2018). Generation of Stable Expression Mammalian Cell Lines Using Lentivirus. Bio-protocol 8(21): e3073. DOI: 10.21769/BioProtoc.3073.
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分类
分子生物学 > DNA > 基因表达
微生物学 > 微生物细胞生物学 > 病毒繁殖
细胞生物学 > 细胞工程 > 慢病毒递送
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