Journal: EBioMedicine

Article Title: A rationally designed nanoparticle for RNA interference therapy in B-lineage lymphoid malignancies

doi: 10.1016/j.ebiom.2014.10.013

Figure Lengend Snippet: Effect of CD22ΔE12 vs. CD22 WT on clonogenicity and self-renewal rate of ALL cells. [A1] Depicted is a representative 1% agarose gel showing the PCR products of FL CD22 and CD22ΔE12–14. [A.2] Subcloning of the CD22 FL or CD22ΔE12–14 genes into the lentiviral pCL6-2AEGwo vector through NheI and XhoI restriction sites was confirmed through restriction enzyme digestion and DNA sequencing. Depicted is a representative 1% agarose gel showing that the generated lentiviral constructs have the correct size FL CD22 (2.5-kb) or CD22ΔE12–14 (2.2-kb) inserts. [B.1] In order to confirm that the lentiviral vectors can be used to achieve expression of FL and truncated CD22 in human cells, 293T cells were transfected with lentiviral constructs for FL CD22, CD22ΔE12–14, as well as pCL6-2AEGwo lentiviral vector without any subcloned CD22. 48 h post transfection cells were examined for FL CD22 and CD22ΔE12–14 mRNA by RT-PCR using the P7, P9, and P10 primer pairs. The P7 primer set was used to amplify a 182-bp region (c.2180–c.2361) of the CD22 cDNA extending from Exon 11 to Exon 13 and spanning the entire Exon 12. The P9 primer set was used amplify a 160-bp region (c.2304–c.2463) of the CD22 cDNA extending from Exon 12 to Exon 14 and spanning the entire Exon 13. The P10 primer set was used to amplify a 213-bp region (c.433–c.645) of Exon 4 of the CD22 cDNA present in both wildtype CD22 and CD22ΔE12–14 mRNA species. As expected all primer sets yielded PCR products in cells transfected with the FL CD22 vector and only the P10 primer set yielded a PCR product in cells transfected with the CD22ΔE12–14 vector. [B.2 and B3] The increased expression levels of the full-length and truncated proteins in transduced 293-T cells (depicted in B.2) and ALL-1 cells (depicted in B.3) were confirmed to be similar by Western blot analysis done at 96 h post-transduction. [C & D] ALL cell lines DAUDI (Burkitt's leukemia/B-ALL) (panels C1 & C.2) and ALL1 (BCR-ABL + B-precursor ALL) (panel C3) were transduced (trans) with wildtype and mutant human CD22 genes using the pCL6-2AEGwo lentiviral vector and then assayed for colony formation in semi-solid methylcellulose cultures without additional stroma support or cytokines. Colony formation was examined using an inverted Nikon Eclipse TS100 microscope with an Epifluorescence attachment. Images were taken using a Digital Sight DS-2MBW Nikon camera (System magnification: 100× or 200 × as indicated). Green fluorescence of colonies resulting from GFP expression confirms successful transduction of the cell lines. [D] Depicted are bar graphs comparing the mean colony numbers in cultures of untransduced and transduced cells.

Article Snippet: The correct PCR products (FL: 2541-bp and ΔE12–14: 2208-bp) were ligated into the 8497-bp lentiviral vector pCL6-2AEGwo through the NheI and XhoI restriction sites (underlined) using the Quick Ligase kit (New England Biolabs catalog no. M2200L) following the manufacturer's instructions.

Techniques: Agarose Gel Electrophoresis, Subcloning, Plasmid Preparation, DNA Sequencing, Generated, Construct, Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transduction, Mutagenesis, Microscopy, Fluorescence