The lentiviral vector pLVSIN-CMV-Neo and Lentiviral High Titer Packaging Mix were obtained from Takara Bio. The lentivirus for expression of FLAG–GPR125 was generated according to the manufacture’s instruction. Briefly, HEK293T cells were cotransfected with pLVSIN-CMV-Neo-FLAG–GPR125 and Lentiviral High Titer Packaging Mix plasmids by using X-tremeGENE HP DNA Transfection Reagent (Sigma–Aldrich). After culture for 48 h, supernatants from transfected HEK293T cells were collected and filtered with a 0.45 μm filter (Millipore). The viral supernatants were added to subconfluent MDCKII cells in the presence of polybrene (2 μg/ml). After 24 h, the virus-containing medium was replaced with fresh DMEM with 10% FCS. The infected cells were cultured for another 24 h and then fixed for immunostaining.
Lentiviral high titer packaging mix
Manufactured by Merck Group
Lentiviral High Titer Packaging Mix is a laboratory product that provides the necessary components for the efficient production of high-titer lentiviral particles. The mix contains the required plasmids and reagents to enable the packaging of lentiviral vectors in a simple and streamlined process.
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2 protocols using lentiviral high titer packaging mix
1
Lentiviral Expression of FLAG-GPR125 in MDCKII Cells
2
Lentiviral Expression of FLAG-TMEM25 in MDCKII Cells
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The lentivirus for expression of FLAG–TMEM25 was generated using pLVSIN-CMV-Neo (Takara Bio) and Lentiviral High Titer Packaging Mix (Takara Bio), according to the manufacture’s instruction. Briefly, HEK293T cells were cotransfected with pLVSIN-CMV-Neo-FLAG–TMEM25 and Lentiviral High Titer Packaging Mix plasmids by using X-tremeGENE HP DNA Transfection Reagent (Sigma–Aldrich). After culture for 72 h, supernatants from transfected HEK293T cells were collected and filtered with a 0.45 μm filter (Millipore). The viral supernatants were added to subconfluent MDCKII cells, and the infected cells were cultured for 24 h. After the virus-containing medium was replaced with fresh DMEM with 10% FCS, the cells were cultured for another 24 h and then seeded on the Transwell chamber for measurement of TER.
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