Journal: Frontiers in Molecular Neuroscience
Article Title: Fyn Signaling Is Compartmentalized to Dopamine D1 Receptor Expressing Neurons in the Dorsal Medial Striatum
Figure Lengend Snippet: Design and characterization FLEX-Cre system. (A,B) Schematic representation of the FLEX vector which encodes a double-Floxed, inverted shRNA and GFP. In the presence of Cre, a FLip-EXcision (FLEX) switch occurs, leading to a U6 promoter-driven expression of the gene-specific shRNA, and a CMV promoter- driven expression of GFP. (C) The FLEX-shFyn plasmid was incubated with (+ Cre) or without (− Cre) Cre recombinase in vitro and infection was evaluated by staining cells with anti-GFP antibodies (right panels). (D,E) Characterization of Cre-driven Fyn knockdown in HEK293T cells. (D,E) FLEX-shFyn or FLEX-SCR plasmids were pretreated with (+ Cre) or without Cre recombinase then co-transfected with Fyn plasmid as indicated. mRNA expression (D) and protein levels (E) were analyzed by RT-PCR and western blot analysis, respectively. 1. pUSE-empty plasmid, 2. pUSE-Fyn, 3. pUSE-Fyn + pLVX-FLEX-SCR, 4. pUSE-Fyn + pLVX-FLEX-SCR + Cre, 5. pUSE-Fyn + pLVX-FLEX-shFyn, 6. pUSE-Fyn + pLVX-FLEX-shFyn + Cre. Two-tailed t -test. *** p
Article Snippet: To differentiate between Fyn expressed in D1R vs. D2R MSNs, we developed a method in which short hairpin RNA (shRNA) that targets a specific gene is expressed only in the presence of Cre-recombinase (Cre).
Techniques: Plasmid Preparation, shRNA, Expressing, Incubation, In Vitro, Infection, Staining, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Two Tailed Test