SARS CoV-2 pseudotyped lentiviruses were produced by transfecting the 293T cells with the pLenti-Puro vectors (Addgene) expressing Luciferase or β-Galactosidase, with pcDNa3.1 vector expressing SARS-CoV-2 spike (BEI repository) and the helper plasmid pSPAX2 (Addgene). The VSV-G and empty lentiviruses were produced by replacing pCDNA3.1-Spike with pcDNA3.1-VSV-G or pCDNA3.1 empty vector, respectively (Addgene). The transfections were carried out using the Polyethylenimine (PEI) method with the ratio at PEI:pLenti:pcNDA3.1-Spike:pSPAX2 = 14:2:2:1 or PEI:pLenti:pVSV-G/pcNDA3.1:pSPAX2 = 10:1:0.5:3. The virus-containing medium was harvested 72 hours after transfection and subsequently pre-cleaned by centrifugation (3,000 g) and a 0.45 μm filtration (Millipore). The virus-containing medium was concentrated by using a LentiX solution (TakaraBio) a 10:1 v/v ratio and centrifuged at the indicated RCF at 4 °C. After centrifugation, the supernatant was carefully removed and the tube was drained on the tissue paper for 3 minutes. Dulbecco’s modified Eagles medium containing 4.5 g/l Glucose (DMEM) was added to the semi-dried tube for re-suspension and then stored at −80 °C.
Pcdna3.1 vsv g
Manufactured by Addgene
pcDNA3.1-VSV-G is a plasmid vector commonly used for gene expression studies. It contains a cytomegalovirus (CMV) promoter, which allows for high-level expression of the gene of interest in mammalian cells. The plasmid also includes the VSV-G (Vesicular Stomatitis Virus Glycoprotein) sequence, which can be used for viral envelope pseudotyping.
Automatically generated - may contain errors
2 protocols using pcdna3.1 vsv g
1
Production of SARS-CoV-2 Pseudotyped Lentiviruses
2
Production of SARS-CoV-2 Pseudotyped Lentiviruses
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SARS CoV-2 pseudotyped lentiviruses were produced by transfecting the 293T cells with the pLenti-Puro vectors (Addgene) expressing Luciferase or β-Galactosidase, with pcDNa3.1 vector expressing SARS-CoV-2 spike (BEI repository) and the helper plasmid pSPAX2 (Addgene). The VSV-G and empty lentiviruses were produced by replacing pCDNA3.1-Spike with pcDNA3.1-VSV-G or pCDNA3.1 empty vector, respectively (Addgene). The transfections were carried out using the polyethylenimine (PEI) method with the ratio at PEI:pLenti:pcNDA3.1-Spike:pSPAX2 = 14:2:2:1 or PEI:pLenti:pVSV-G/pcNDA3.1:pSPAX2 = 10:1:0.5:3. The virus-containing medium was harvested 72 h after transfection and subsequently pre-cleaned by centrifugation (3,000g at 4°C) and a 0.45 μm filtration (Millipore). The virus-containing medium was concentrated by using a LentiX solution (TakaraBio) a 10:1 v/v ratio and centrifuged at the indicated RCF at 4°C for 45 min. After centrifugation, the supernatant was carefully removed, and the tube was drained on the tissue paper for 3 min. Dulbecco’s modified Eagle’s medium containing 4.5 g/L glucose (DMEM) was added to the semi-dried tube for re-suspension and then stored at −80°C.
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