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Method Article
Producing Lentivirus in 293T Cells with Polyjet
Xiaoliang Leon Xu
Xiaoliang Leon Xu, Memorial Sloan-Kettering Cancer Center
Corresponding Author
David Cobrinik
David Cobrinik, Children's Hospital Los Angeles
Corresponding Author
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
Lentiviruses were produced by reverse transfection of suspended 293T cells using 20 ug lentiviral vector, 10 ug pVSV-G, 20 ug pCMV-dR8.91, and 100 μl Polyjet (SignaGen) or Lipofectamine 2000 (Life Technologies) in 15 cm dishes. The 3 ml plasmids–Polyjet complex and 1.5 ml 293T cell suspension were mixed in 50 ml centrifuge tubes and shaken for half hour before transferred to dishes. Virus harvested 48 and 72 h after transfection was combined, concentrated 50-100-fold by centrifugation at 26,000 rpm for 90 minutes, and suspended in culture medium. 500-2,000 μl of concentrated virus was used to infect 1 x 10(6) cells in 500 μl culture medium in the presence of 4 μg/ml polybrene (Sigma-Aldrich) followed by gentle pipetting 25 times and shaking for 10 minutes in the hood.
Keywords
Lentivirus; Production; Polyjet; 293T; Infection
Xiaoliang Leon Xu
commented on 13 November, 2014
BIOWORLD CELLSTAR TISSUE CULTURE DISHES (Catalog No.: 50-255-154) should be used for transfection of 293T cells in this lentivirus production protocol. Leon Xu
Xiaoliang Leon Xu
commented on 13 November, 2014
BIOWORLD CELLSTAR TISSUE CULTURE DISHES (Catalog No.: 50-255-154) should be used for transfection of 293T cells in this lentivirus production protocol. Leon Xu
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Associated Publications
Retinoblastoma Has Properties of a Cone Precursor Tumor and Depends Upon Cone-Specific MDM2 Signaling
by Xiaoliang L. Xu, Yuqiang Fang, Thomas C. Lee, +7
Cell (13 September, 2014)
Rb suppresses human cone-precursor-derived retinoblastoma tumours
by Xiaoliang L. Xu, Hardeep P. Singh, Lu Wang, +5
Nature (29 September, 2014)
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This is a preprint. It has not completed peer review.
Method Article
Producing Lentivirus in 293T Cells with Polyjet
Xiaoliang Leon Xu
Xiaoliang Leon Xu, Memorial Sloan-Kettering Cancer Center
Corresponding Author
David Cobrinik
David Cobrinik, Children's Hospital Los Angeles
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 29 Sep, 2014
Community comments: 2
Metrics
Comments: 2
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
Lentiviruses were produced by reverse transfection of suspended 293T cells using 20 ug lentiviral vector, 10 ug pVSV-G, 20 ug pCMV-dR8.91, and 100 μl Polyjet (SignaGen) or Lipofectamine 2000 (Life Technologies) in 15 cm dishes. The 3 ml plasmids–Polyjet complex and 1.5 ml 293T cell suspension were mixed in 50 ml centrifuge tubes and shaken for half hour before transferred to dishes. Virus harvested 48 and 72 h after transfection was combined, concentrated 50-100-fold by centrifugation at 26,000 rpm for 90 minutes, and suspended in culture medium. 500-2,000 μl of concentrated virus was used to infect 1 x 10(6) cells in 500 μl culture medium in the presence of 4 μg/ml polybrene (Sigma-Aldrich) followed by gentle pipetting 25 times and shaking for 10 minutes in the hood.
Xiaoliang Leon Xu
commented on 13 November, 2014
BIOWORLD CELLSTAR TISSUE CULTURE DISHES (Catalog No.: 50-255-154) should be used for transfection of 293T cells in this lentivirus production protocol. Leon Xu
Xiaoliang Leon Xu
commented on 13 November, 2014
BIOWORLD CELLSTAR TISSUE CULTURE DISHES (Catalog No.: 50-255-154) should be used for transfection of 293T cells in this lentivirus production protocol. Leon Xu
4 comments flagged for moderation
If you posted a comment and think it was mistakenly removed, please contact customer support at [email protected].
Associated Publications
Retinoblastoma Has Properties of a Cone Precursor Tumor and Depends Upon Cone-Specific MDM2 Signaling
by Xiaoliang L. Xu, Yuqiang Fang, Thomas C. Lee, +7
Cell (13 September, 2014)
Rb suppresses human cone-precursor-derived retinoblastoma tumours
by Xiaoliang L. Xu, Hardeep P. Singh, Lu Wang, +5
Nature (29 September, 2014)