A DNA plasmid containing Katushka2S (K2S) was synthesized in a pUC57-Kan backbone (GenScript). The Katushka2S sequence was cloned into a lentiviral construct containing CD63 (LV112335, Applied Biological Materials) so that K2S is fused to CD63 on the C-terminus of CD63. D1 MSCs were transduced with lentivirus containing the CD63-K2S plasmid using standard techniques [31 ]. Briefly, lentiviral particles were produced with a 2nd generation lentiviral packaging system (LV003, Applied Biological Materials) using Lentifectin (Applied Biological Materials) in HEK293T cells. Lentiviral particles were purified and applied to D1 MSCs at passage 10 with 8 μg/mL polybrene (Sigma) for 3 days. Cells were expanded over a period of several days to reach ~80% confluency. Then, cells were sorted using a MoFlo Astrios (Beckman Coulter) based on their CD63-K2S signal compared to non-transduced cells of the same passage. Concentrated EV solutions were shown to be positive for CD63-K2S versus EVs from non-transduced cells using IVIS imaging (Living Image 4.0, Perkin Elmer).
Puc57 kan backbone
Manufactured by GenScript
The PUC57-Kan backbone is a plasmid vector commonly used in molecular biology and genetic engineering. It contains a kanamycin resistance gene for selection in bacterial hosts, as well as a multiple cloning site for the insertion of foreign DNA sequences.
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2 protocols using puc57 kan backbone
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Engineered Extracellular Vesicles Tracking
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Engineered Extracellular Vesicles Tracking
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A DNA plasmid containing Katushka2S (K2S) was synthesized in a pUC57-Kan backbone (GenScript). The Katushka2S sequence was cloned into a lentiviral construct containing CD63 (LV112335, Applied Biological Materials) so that K2S is fused to CD63 on the C-terminus of CD63. D1 MSCs were transduced with lentivirus containing the CD63-K2S plasmid using standard techniques [31 ]. Briefly, lentiviral particles were produced with a 2nd generation lentiviral packaging system (LV003, Applied Biological Materials) using Lentifectin (Applied Biological Materials) in HEK293T cells. Lentiviral particles were purified and applied to D1 MSCs at passage 10 with 8 μg/mL polybrene (Sigma) for 3 days. Cells were expanded over a period of several days to reach ~80% confluency. Then, cells were sorted using a MoFlo Astrios (Beckman Coulter) based on their CD63-K2S signal compared to non-transduced cells of the same passage. Concentrated EV solutions were shown to be positive for CD63-K2S versus EVs from non-transduced cells using IVIS imaging (Living Image 4.0, Perkin Elmer).
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