psin ef1 puro vector  (Addgene inc)

Journal: Advanced Science

Article Title: Functional Immune Cell‐Derived Exosomes Engineered for the Trilogy of Radiotherapy Sensitization

doi: 10.1002/advs.202106031

Figure Lengend Snippet: Fabrication and characterization of engineered M1Exos. A) The gene structure of lentiviral vector coding the fusion protein CAT‐TMR‐Anti‐PD‐L1‐c‐Myc, and its expression pattern on cell membrane of RAW 264.7 cells. Anti‐PD‐L1‐c‐Myc was located on the outside of cell membrane, and CAT was on the inside. This structure was inspired by Car‐T cell. TMR, transmembrane region. Signal P, signal peptide. B) The expression of the fusion protein in RAW 264.7 was examined by immunofluorescence staining of c‐Myc tag. The fusion protein could be successfully expressed on cell membrane after stable transfection of lentivirus (+). Nucleus was stained with DAPI. Scale bar, 20 µm. C) The stable expression of fusion protein on cell membrane of RAW 264.7 was confirmed by flow cytometry. Gray, blank RAW 264.7 cells without staining; blue, cells were stained with Alexa Fluor 647 (AF647)‐conjugated anti‐rabbit secondary antibodies; red, cells were first stained with rabbit anti‐c‐Myc antibodies, followed by the staining of secondary antibodies. D) TEM images of M1Exos (1), CAT‐PD‐M1Exos (2), and DDRi@CAT‐PD‐M1Exos (3). Scale bar, 100 µm. E) SEM images of DDRi@CAT‐PD‐M1Exos. Scale bar, 100 µm. F) The hydrodynamic diameter distribution and G) zeta potentials of 1) M1Exos, 2) CAT‐PD‐M1Exos, and 3) DDRi@CAT‐PD‐M1Exos. H) The co‐localization of the anti‐c‐Myc antibody‐stained fusion protein and the DiI‐labeled DDRi@CAT‐PD‐M1Exos by CLSM, confirming the existence of fusion protein on DDRi@CAT‐PD‐M1Exos. Scale bar, 1.0 µm. I) The affinity between Anti‐PD‐L1 on DDRi@CAT‐PD‐M1Exos and mouse PD‐L1 protein was verified by dot blotting. PD‐L1 protein was adsorbed on nitrocellulose membrane, followed by the addition of PBS (I), DDRi@CAT‐M1Exos (II), CAT‐PD‐M1Exos (III), DDRi@CAT‐PD‐M1Exos (IV), DDRi@CAT‐PD‐M1Exos plus excess amount of Anti‐PD‐L1 (V), and the binding of different M1Exos onto membrane was examined by HRP (horse radish peroxidase)‐conjugated anti‐c‐Myc. The expression of CD45 on M1Exos was examined as input, and the equal amounts of different M1Exos (0.5 µg) were adsorbed onto membrane, followed by the addition of HRP‐conjugated antimouse CD45. J,K) The affinity between Anti‐PD‐L1 (on DDRi@CAT‐PD‐M1Exos) and PD‐L1 (on LLC cells) was verified by J) flow cytometry and K) CLSM. LLC cells were incubated with DiI‐labeled CAT‐M1Exos (II), DDRi@CAT‐PD‐M1Exos (IV), and DDRi@CAT‐PD‐M1Exos plus excess amount of Anti‐PD‐L1 (V). Nucleus was stained with DAPI. Scale bar, 30 µm. (L) The competition binding assay for confirming the affinity of Anti‐PD‐L1 on DDRi@CAT‐PD‐M1Exos. LLC cells were co‐incubated with a fixed concentration of Cy5.5‐conjugated Anit‐PD‐L1 along with gradient concentrations of DDRi@CAT‐M1Exos or DDRi@CAT‐PD‐M1Exos. The concentration of un‐binding Cy5.5‐Anit‐PD‐L1 was measured by fluorescence intensity of Cy5.5. (M) Confirmation of enzyme activity of CAT in DDRi@CAT‐PD‐M1Exos. The concentration of H 2 O 2 was measured by the ammonium molybdate method, and the decrease of absorbance at 405 nm indicating the decomposition of H 2 O 2 . (N) The relative enzyme activity of CAT after incubating free CAT, DDRi@CAT‐M1Exos or DDRi@CAT‐PD‐M1Exos with proteinase K for different time (0–12 h). The membrane of M1Exos could protect CAT from hydrolysis. O) DDRi@CAT‐PD‐M1Exos was mixed with different concentration of H 2 O 2 (6.25–50 × 10 −6 m ), and the concentration of dissolved oxygen was measured by on‐line oxygen dissolving meter. The control group was 50 × 10 −6 m H 2 O 2 without the addition of DDRi@CAT‐PD‐M1Exos. P,Q) Hypoxia level of LLC cells after incubating cells with a) PBS, b) DDRi@M1Exos, c) DDRi@CAT‐PD‐M1Exos and d) free CAT. Cells were maintained in hypoxia condition with 10 × 10 −6 m H 2 O 2 . Cellular hypoxia was stained with the hypoxyprobe‐1 kit, and nucleus was stained with DAPI. Data are presented as mean ± SD ( n = 5, * p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Using restriction enzyme digestion and digation, the sequence was cloned into the EcoRI and MluI sites of lentiviral vector pSIN4‐EF1a‐LMO2‐IRES‐Puro (Addgene, USA, Plasmid #61064).

Techniques: Plasmid Preparation, Expressing, Immunofluorescence, Staining, Stable Transfection, Flow Cytometry, Labeling, Binding Assay, Incubation, Concentration Assay, Fluorescence, Activity Assay