recipient vector pcdna flag mzscan4d polya  (Addgene inc)

Journal: Pharmaceuticals

Article Title: Brain-Wide Transgene Expression in Mice by Systemic Injection of Genetically Engineered Exosomes: CAP-Exosomes

doi: 10.3390/ph17030270

Figure Lengend Snippet: Generation and characterization of genetically engineered MSCs-derived exosomes. ( A ). The schematic diagram showing the various steps including isolation and growth of BM-MSCs derived-AAV-CAP domain-Lamp2b fusion protein expression plasmid recombination, generation of BM-MSC derived CAP-Exosomes and Lamp2b-exosomes. ( B ). Western blot analysis shows expression of fusion protein CAP-Lamp2b-HA in total protein isolated 48 h after electroporation of CAP-Lamp2b with BM-MSCs as both the Hemagglutinin HA-tag antibody specific and Lamp2b- specific positive signal shows in the 1st western blot run (( B ). Right panel in lane1) and after striping of 2nd western blot (( B ), Left panel, lane1) respectively, and neither in the protein isolated from pcDNA-hygro-Lamp2b electroporated BM-MSCs (( B ), Lane 2, Right and Left panel), nor in the total protein isolated from just BM-MSCs (( B ), Lane 3, Right and Left panel). ( C ). Western blot analysis of a total of 10 9 exosomal particles (average size of 100–150 nm) showed that both Lamp2b and HA specific signal are present in ( C ) (Lane 2) indicating the CAP-Lamp2b-HA fusion protein inclusion on the exosomes.

Article Snippet: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ph17030270/s1 , Figure S1: Graphical presentation of cloning strategy for AAV-CAP domain in pcDNA GNSTM-3-Flag-10-lamp2b-HA expression plasmid vector. (Addgene, USA, Plasmid no.71293).

Techniques: Derivative Assay, Isolation, Expressing, Plasmid Preparation, Western Blot, Electroporation

Journal: Pharmaceuticals

Article Title: Brain-Wide Transgene Expression in Mice by Systemic Injection of Genetically Engineered Exosomes: CAP-Exosomes

doi: 10.3390/ph17030270

Figure Lengend Snippet: Targeting efficiency and specificity of CAP-Exosomes and Lamp2b-exosomes after systemic delivery. ( A ) a schematic for the production of CAP-exosomes and Lamp-2b-exosomes. Presence of high numbers of DiI-labeled (red) CAP-exosomes ( B ) compared to Lamp2b-exosmes ( C ) in the frontal cortex region of respective mice. In the frontal cortex (FC) of the mice very high DiI-labeled exosomes were found (3D-confocal micrograph ( D ), red fluorescence) along with high efficiency GFP-transgene expression (( E ), green fluorescence). Note that visual inspection of merging (Red + Green) confocal micrograph ( F ) revealed a high percentage of only red fluorescence but not merged colors. NeuN stained micrograph of the hippocampus (( G ), white) with DiI labeled exosomes (( H ), red) and GFP- expression (( I ), green) indicate that systemically delivered CAP-Exosomes loaded GFP- transgene capable of brain wide and highly neuron specific delivery of genes and its expression in the mouse brain. Both the red ( J ) and green ( K ) fluorescence were low indicating CAP- domain expressing exosomes minimizing off-target GFP-gene expression. In a separate sets of animals IV injection of DiI labeled and GFP loaded hygro-Lamp2b exosomes to mice resulted in high exosomal uptake ( L ) and GFP-gene expression ( M ) in the animal’s liver.

Article Snippet: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ph17030270/s1 , Figure S1: Graphical presentation of cloning strategy for AAV-CAP domain in pcDNA GNSTM-3-Flag-10-lamp2b-HA expression plasmid vector. (Addgene, USA, Plasmid no.71293).

Techniques: Labeling, Fluorescence, Expressing, Staining, IV Injection