Lentiviral Transduction of T-ALL and Cord Blood

Lentiviral vectors (pLV-shRNA-EGFP or mCherry:T2A:Puro-U6) was purchased (VectorBuilder) and wild-type and mutant ADAR1^E912A (pCDH-EF1-T2A-copGFP) were produced according to published protocol ^{25 (link)}. All lentivirus was titer by transduction of HEK293T cells and efficiency was assessed by p24 ELISA and RT-qPCR of the 5’ LTR region. Lentiviral transduction of primary T-ALL or cord blood samples was performed at a MOI of 100–200. The cells were cultured for 3–4 days in 96-well plate (2X10⁵-5X10⁵ cells per well) containing StemPro (Life Technologies) media supplemented with human IL-6, stem cell factor (SCF), Thrombopoietin (TPO) and FLT3 (all from R&D Systems). For T-ALL cell lines, the cells were transduced at a MOI of 20–50 in culture media.

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Rivera M., Zhang H., Pham J., Isquith J., Zhou Q.J., Sasik R., Mark A., Ma W., Holm F., Fisch K.M., Kuo D.J., Jamieson C, & Jiang Q. (2023). Malignant A-to-I RNA editing by ADAR1 drives T-cell acute lymphoblastic leukemia relapse via attenuating dsRNA sensing. Research Square.

Publication Preprint 2023

pLV-shRNA-EGFP mCherry:T2A:Puro-U6 human IL-6 Thrombopoietin (TPO)

Cell lines Cells Cord blood Culture media Elisa Flt3 Human Lentivirus Shrna Stem cell factor T all Thrombopoietin Vectors

Corresponding Organization :

Other organizations : University of California, San Diego, Karolinska Institutet

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Variable analysis

independent variables

Lentiviral vectors (pLV-shRNA-EGFP or mCherry:T2A:Puro-U6)
Wild-type and mutant ADAR1^E912A (pCDH-EF1-T2A-copGFP)

dependent variables

Transduction efficiency assessed by p24 ELISA and RT-qPCR of the 5' LTR region
Proliferation or other phenotypic changes in primary T-ALL or cord blood samples, and T-ALL cell lines

control variables

Multiplicity of infection (MOI) of 100-200 for primary T-ALL or cord blood samples, and 20-50 for T-ALL cell lines
Culture duration of 3-4 days in 96-well plate with 2X10^5-5X10^5 cells per well
StemPro media supplemented with human IL-6, stem cell factor (SCF), Thrombopoietin (TPO) and FLT3 for primary T-ALL or cord blood samples
Culture media for T-ALL cell lines

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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