Reducing BRCA1 Protein in Mouse Neurons

Six anti-Brca1 shRNAs were obtained from Sigma-Aldrich (Mission shRNA lentiviral particles, parental vector on pLKO.1-purobackbone). Their ability to reduce BRCA1 protein levels was assessed in primary mouse neuronal cultures 1 week after infection. The sequences of the two most effective shRNAs (sh1 and sh2) were: sh1: 5′-CCACAGGTAAATCAGGAATTT-3′ (ref. 15 (link)) and sh2: 5′-GTGCTTCCACACCCTACTTAC-3′. The sh1 sequence is in the coding sequence of BRCA1 mRNA, and the sh2 sequence is in the 3′-untranslated region. The sequence of the control, scrambled shRNA was 5′-CCACTACCGTTGTTATAGGTG-3′. Each sequence was inserted into the FUGW plasmid backbone and used to generate lentiviral vectors as described6 (link)62 (link). Lentiviruses were bilaterally injected stereotaxically into the DG as described6 (link), using 1.5 × 10⁶ titre unit (TU) per DG. No specific method of randomization was used to determine the treatment of each mouse. GFP expression was used as an indication of viral transduction. Note, however, that the GFP intensity does not reliably reflect the abundance of shRNA expressed in transduced cells, as the promoters driving the expression of these transgenes are activated by different RNA polymerases: GFP expression is RNA polymerase II dependent, whereas expression of the shRNA is RNA polymerase III dependent.