Author: Banu, Nasirah; Chia, Adeline; Ho, Zi Zong; Garcia, Alfonso Tan; Paravasivam, Komathi; Grotenbreg, Gijsbert M.; Bertoletti, Antonio; Gehring, Adam J.
Title: Building and Optimizing a Virus-specific T Cell Receptor Library for Targeted Immunotherapy in Viral Infections Document date: 2014_2_25
ID: 44w6omdp_30
Snippet: TCR introduction into primary T cells. Retroviral vector, MP71, was used to deliver the TCR genes into the primary human T cells. 2 3 10 6 Phoenix amphotropic packaging cells were seeded into 100 mm tissue culture dishes 24 h prior to transfection. Phoenix cells were transiently co-transfected using calcium phosphate with 9 mg each of MP71-TCR Va, MP71-TCR Vb together with 6 mg of amphotropic envelope (PCL-Amp) for 24 h. For P2A-linked constructs.....
Document: TCR introduction into primary T cells. Retroviral vector, MP71, was used to deliver the TCR genes into the primary human T cells. 2 3 10 6 Phoenix amphotropic packaging cells were seeded into 100 mm tissue culture dishes 24 h prior to transfection. Phoenix cells were transiently co-transfected using calcium phosphate with 9 mg each of MP71-TCR Va, MP71-TCR Vb together with 6 mg of amphotropic envelope (PCL-Amp) for 24 h. For P2A-linked constructs, 18 mg of the TCR plasmid was co-transfected with 6 mg of the amphotropic envelope. After 24 h, IMDM was replaced with Aim-V supplemented with 2% human AB serum and phoenix cells were incubated for an additional 24 h before retroviral supernatants were collected for transduction.
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