Chunk #40 — EXPERIMENTAL PROCEDURES — In vivo birth-dating, genetic manipulation of neural progenitors with engineered retroviruses and pharmacological treatments
Engineered self-inactivating murine oncoretroviruses were used to co-express shRNA under the U6 promoter and GFP under the EF1α promoter (pUEG vector), or to co-express a transgene under the Ubiquitin promoter and GFP under IRES sequence (pCUXIE vector) specifically in proliferating cells and their progeny. Specific shRNAs against mouse disc1 (DISC1-shRNA) (Duan et al., 2007) and mouse pten (PTEN-shRNA) (Jaworski et al., 2005) have been characterized previously and were further confirmed their specificity and efficacy in knocking down endogenous proteins in hippocampal neurons using lentiviruses. Three different shRNAs against mouse KIAA1212 were cloned into the pUEG vector with the following sequences: shRNA-K1: 5’- CGTTACAATCAGTTATTAA -3’; shRNA-K2: 5’- CACTAGCAA TAGCAATAAT -3’; shRNA-K3: 5’- GAAGGAGAG GCAACTGGAT -3’. The efficacy of knocking down endogenous KIAA1212 was confirmed in hippocampal neurons using lentiviruses. The shRNA-K3 sequence is identical to the one previously characterized (Kitamura et al., 2008). High titers of engineered retroviruses were produced by co-transfection of retroviral vectors and vesicular stomatitis viral envelope into 293gp cells followed by ultra-centrifugation of viral supernatant as previously described (Duan et al., 2007).
