prior to sacrifice (for P2 animals, BrdU was injected in pregnant dams half day prior to give birth). Immunohistochemistry was carried on as described above using a rat anti-CldU/BrdU primary antibody (1:200, monoclonal, Immunologicalsdirect, Oxfordshire, UK), with an additional denaturation in 1 M HCl for 30 minutes prior to pre-incubation with serum. To assess the neuronal-inducing activity of Ngn2 and NeuroD1, transduced cultures were incubated with CldU (20 mM, Sigma) for 2 days, after a period of differentiation of five days. For immunocytochemistry, cultures were fixed with 4% paraformal- dehyde at day 7, rinsed with PBS three times, treated with 1 M HCl at 65uC for 5–10 min, pre-incubated and then incubated with a rat anti-CldU/BrdU antibody and other primary antibodies. The remainder of the procedure was performed according to the protocol for immunohistochemistry already mentioned. In utero surgery Timed pregnant female Sprague Dawley rats with embryos at gestational age E15.5 were anesthetized with halothane. The mother was placed in the lower level of a two-level wooden stage. The abdomen was shaved with an electric razor and then cleaned with 70% alcohol. A 2–3 cm midline laparotomy was performed. Each uterine horn was carefully taken out individually and the number of embryos recorded. One horn was then placed back inside the mother, whilst the other horn was prepared for injection. The embryos were kept moist with constant application of warm saline to prevent dehydration. Approximately 2 ml of viral suspension (1610‘9TU/ml) was injected into the lateral ventricle of each embryo, except for the embryo closest to the vagina. After the injections, the uterine horns were placed back into the abdomen. The abdominal wall and the overlying skin were then sutured. Care was taken not to damage abdominal muscles so that normal delivery of the pups was possible at term. The entire surgery generally took about 45 minutes to one hour. Each mother was allowed to recover in her cage before being returned to the animal stable. Shredded paper was added to each cage to encourage nesting and special care was taken not to stress the mothers. Following normal delivery, the pups were allowed to develop to adulthood up to two weeks. Quantification For the in vivo experiment, manual cell counting was performed on 18 and 30 mm thick brain sections for two days and two weeks old animals, respectively. The brain of P2 animals was cut into 10 series of coronal sections; the brain of two-weeks old animals was cut into 7–8 series of coronal sections. For each staining, one-two series from three to five different individuals were analyzed per genotype using a confocal microscope with 1, 2 or 3 lines of excitation, in sequential scanning in order to avoid false positives. When two series were analyzed, only one series was counted. The mean number of cells per hippocampus expressing the markers of interest, was calculated for each brain, based on the analysis of 6–8 consecutive dorsal hippocampal sections. The final mean number of cells per section was calculated by adding the mean number from each individual. We expressed the data as the mean number of positive cells6standard error of the mean (SEM). In this study, more than 25 Ngn2 null, .45 Ngn2 heterozygotes, and .60 WT individuals (including rats injected with retroviruses) were analyzed, in total. Statistical comparison was performed using one-factor analysis of variance (ANOVA) with transcription factor, cellular marker, genotype or time as variables, followed by post- hoc analysis when significant differences were observed, using Statview 5.0 software (SAS institute Inc.). For cell culture experiments, three independent experiments were performed, Hippocampal Neurogenesis each in duplicate. The counting was based on seven randomly chosen different fields of view. For each diagram, the level of significance (p-value) is represented as follows: P,0.05 = *; P,0.001 = **; P,0.0001 = ***. All data are expressed as6stan- dard error of the mean (SEM). Human sample and RT-PCR The brain from a 64 year-old male was provided by the Harvard Brain Tissue Resource Center. The individual whose brain tissue was being analyzed gave written consent for storage and use of his tissue for research. Five millimeters thick fresh human hippocampal tissue sample was snap frozen. The frozen tissue block was cut into 20 series of coronal sections, using a cryostat. Every 10 sections, one section was placed in an Eppendorf tube, on dry ice. Pooled sections were used for RT-PCR. Total RNA was prepared using Trizol and RNAeasy, supplemented with RNAGuard as previously described [49]. The RNA was digested with shrimp DNAse before cDNA synthesis, which was performed using a mix of oligo-dT and random hexamer primers and SuperscriptII reverse transcriptase. Advantage2 polymerase mix (Clontech/BRL) was used for PCR, with the following cycling conditions: 10 cycles of 94u C for 30 seconds, 68uC for 2 minutes, 30 cycles of 94uC for 30 seconds, 60uC for 1 minute, 68uC for 1 minute and 30 seconds, one cycle of 68u C for 2 minutes, soak at 16uC. Primers marked * were designed by PrimerBank. hRPS18 (sense) 59-GCCTTTGCCATCACTGCCATT and hRPS18 (antisense) 59-GCCAGTGGTCTTGGTGTGCT, hPAX6 (sense) 59-GCCCTGGAGAAAGAGTTTGAGAGAACCCATT hPAX6 (antisense) 59-GGGGAAATGAGTCCTGTTGAAGT- GGTGC, hTBR1 (sense) 59-GCGGACACCAATGTGCAAG- GAAATCG and hTBR1 (antisense) 59-CGAGGGGGTCAGG- CGGTCCATGTCACAGC, hTBR2 (sense) 59-GACC- TGTGGCAAAGCCGACAATAACATGC and hTBR2 (antisense) 59-GGGGGTGTCTCTATCCAAGAAGAGCCAAT, hPDHX ( = PROX1) (sense) 59-GGGACACTACGGTTCCGTTTAAGTC- CAGC and hPDHX (antisense) 59-CTCTCCATCCCAGCTTA- CATTAACATCTGGCATTTGT, hNGN2 (sense) * 59-CATCAA- GAAGACCCGTAGACTGA and hNGN2 (antisense)* 59- CAACACTGCCTCGGAGAAG, hMASH1 (sense) 59- CCTGGATCCGCATGGAAAGCTCTGCCAAGATGGAG and hMASH1 (antisense) 59-CCTGGATCCCCCCTCAGAACCAG- TTGGTGAAGTCGA, hNEUROD1 (sense) 59-GCTCAGGACC- TACTAACAACAAAGGAAATCGAAACATG and hNEUROD1 (antisense) 59-CAAAGCGTCTGAACGAAGGAGACCAGGT, hGLAST (sense) 59-CATCAGGGAAGATGGGAATGC- GAGCTGTAGTCTATTAT and hGLAST (antisense) 59- CCACGGGGGCATACCACATTATTACTGCTACC, hNEST (sense) 59-CAGGAGCGGCTGCGGGCTACTGAAAAGTTCC and hNEST (antisense) 59-CAGGGCTGAGGGGTGGTGC- CAAGGAGG, hGFAP (sense) 59-CCACGAGGAGGAGGTTC- GGGAACTCCAGGAGC and hGFAP (antisense) 5-GGAAT- GGTGATCCGGTTCTCCTCGCCCTCTAGC, hCALB2 ( = Calretinin) (sense) 59-GGCTCTGGCATGATGTCAAAGAGT- GACAACTTT and hCALB2 (antisense) 59-GGGCATCCAGCT- CATGCTCGTCAATGTAGCC, hCALB1 (sense) 59-GCGAAA- GAAGGCTGGATTGGAGTTATCACC and hCALB1 (antisense) 59-CCCTCCATCCGACAAAGCCATTATGTTCTTCTTGTA- TG, hSOX2 (sense) 59-GGAGAACCCCAAGATGCACAACTC- GGAGAT and hSOX2 (antisense) 59-GAGGAAGAGGTAACCA- CAGGGGGGCTGGAGC, hWNT3A (sense) 59-CCGAGGG- CATCAAGATTGGCATCCAGGAGTGC and hWNT3A (anti- sense) 59-TCGGGTTGCGACCACCAGCATGTCTTCACCTC, hSHH* (sense) 59-ACTCCGAGCGATTTAAGGAACT and hSHH* (antisense) 59-CAGACGTGGTGATGTCCACTG. PLoS ONE | www.plosone.org 16 March 2009 | Volume 4 | Issue 3 | e4779