Figure 1.

(A) Generation of ARMS shRNA-GFP expressing lentivirus. The ARMS shRNA cassette was subcloned into a plasmid (pBluescript) downstream of the human U6 RNA polymerase III promoter. The plasmid region containing the cassette and the promoter was then subcloned into the FG12 lentiviral vector, which also encodes for GFP under the control of Ubiquitin C (UbiC) promoter. (B) Analysis of the efficiency of lentiviral transduction of primary cortical neurons. Primary cortical neurons were transduced with ARMS shRNA-GFP lentivirus (MOI 10) as outlined in Materials and Methods. Panel on the left-hand indicates GFP-positive cells, whereas the right-hand panel shows Hoechst 33342 staining of the same microscopic field (C) Analysis of ARMS expression following lentivirus infection. Cell lysates were prepared from infected cortical neurons and immunoblot analyses were performed using antibodies specific to ARMS and α-tubulin. ARMS expression was reduced by approximately 80%. (D) Functional validation of ARMS shRNA-GFP expressing lentivirus. Primary cortical neurons were infected with indicated lentiviruses for 7 days. On DIV9, 10μM of FM1-43 was added to the medium for 15 minutes. After washing, the cells were resuspended in HBSS plus 100mM KCl for depolarization. The release of FM1-43 was monitored and recorded over 60 minutes at 1 minute intervals. As previously reported (Cortes et al., 2007), synaptic activity was increased in the absence of ARMS.