Cerebellum (2010) 9:537–547 N-myc and c-myc. Primer sequences were as follow: Cre1: GCC TGC ATT ACC GGT CGA TGC AAC GA; Cre2: GTG GCA GAT GGC GCG GCA ACA ACC ATT; N-myc1: GTC GCG CTA GTA AGA GCT GAG ATC; N-myc2: GGC ACA CAC CTA TAA TCC CAG CTA; N-myc3: CAC AGC TCT GGA AGG TGG GAG AAA GTT GAG CGT CTC C; c- myc1: GCC CCT GAA TTG CTA GGA AGA CTG; c-myc2: CCG ACC GGG TCC GAG TCC CTA TT. Cre1 and 2 primer set amplified a 700 bp band from Cre insertion. The flox-specific band for N-myc1, 2, and 3 primers is 260 bp, the wild-type band is 217 bp, and the deletion band is 350 bp. The flox-specific band for c-myc1 and 2 primer set is 500 bp, and the wild-type band is 400 bp. A separate primer set to detect c-myc deletion, c-mycDS: TCG CGC CCC TGA ATT GCT AGG AA, and c-mycDA: TGC CCA GAT AGG GAG CTG TGA TAC TT, were used and amplifies a band at ~750 bp. Immunohisto- and Cytochemistry All E12.5 embryos were immersion-fixed overnight in fresh, buffered 4% paraformaldehyde, paraffin embedded and cut (12 μm) sagittally. Pregnant female were anesthetized with 150 mg/kg ketamine and 16 mg/kg xylazine, then E17.5 embryos were removed individual- ly and perfused with buffered 4% paraformaldehyde. Brains were removed and fixed overnight in fresh, buffered 4% paraformaldehyde, then cryopreserved and cut (12 μm) sagittally. For immunostaining, E12.5 embryo sections were deparaffinized then rehydrated and 30 mM sodium citrate treated for 10 min at 95°C, while E17.5 frozen brain sections were post-fixed in −20°C Acetone for 10 min followed by three 5 min PBS wash. All sections were blocked in 10% normal goat serum in PBS for 1 h at room temperature then incubated in primary antibody overnight at 4°C. Anti- bodies used include anti-BrdU (Chemicon MAB3424, 1:150), anti-PhosphoH3 (Upstate 06-570, 1:200), and anti-TubulinßIII (TUJ; Covance PRB-435P, 1:150). The next day, sections were washed in PBS three times 10 min each, and then incubated in secondary antibody for 2 h at room temperature. Alexa Fluor Goat α Rabbit IgG λ488 (Invitrogen A11008) and Alexa Fluor Goat α Mouse IgG λ546 (Invitrogen A 11003) were used at 1:1,000 dilution. Sections were washed again in PBS three times 10 min each, and then mounted in Vectashield mounting medium with DAPI (VECTOR H- 1200). Anti-BrdU staining was conducted on embryos from pregnant females who were IP injected with BrdU (Sigma B5002, 150 μg/g) following a 2–4 h BrdU incorporation period. TUNEL staining was performed using the DeadEnd Fluorometric TUNEL System Kit (Promega G3250) as directed. 539 Microarray and Quantitative Real-Time PCR Cortex, midbrain and cerebellum were dissected from brains of freshly sacrificed E17.5 embryos. Total RNA was isolated from these brain areas using RNeasy Mini Kit (Qiagen 74134) with DNaseI digestion (Invitrogen 18068- 015) performed post-RNA extraction. Quality of RNA was checked and cRNA was produced by UC Davis Gene Expression Analysis facility for hybridization to Sentrix Mouse Ref-8 Expression microarray. qPCR was performed on total RNA from the same batch sent off for array. Samples were processed using Express SYBR Green qPCR Supermix (Invitrogen A10314) according to manufacture protocol. All qPCR data were generated using ß-Actin as reference (house-keeping) gene. This gene was chosen based on microarray results, which showed no significant expression variation between control and double knockout brains. Primer efficiency was tested by generating standard curves for all primers against all brain areas. Cycling was conducted as follows: pre-incubation at 95°C for 5 min, 45 cycles of amplifica- tion at 95°C for 10 s, 60°C for 20 s, and 72°C for 30 s, followed by one cycle of melting curve at 95°C for 5 s, 65°C for 1 min, and 97°C continuous. Results Double Knockout of c- and N-myc in NSC Severely Disrupts Brain and Cerebellar Growth, Resulting in Lethality as Early as E17.5 Previous separate single conditional targeted disruption of either c- or N-myc in NSC using nestin-cre caused varying degrees of impaired brain growth manifesting as microcephaly, however even in the case of N-myc, which had a >40% reduction in brain growth, there was no lethality apparent [16]. In those studies, an underrepresen- tation of N-myc flox/flox nestin-cre (NSC specific N-myc knockout) mice was present in breedings of N-myc flox/ flox X N-myc flox/WT nestin-cre mice, but equally underrepresented were N-myc flox/WT cre-negative (healthy, non-phenotypic) offspring, indicating that there was likely a genetic linkage between the N-myc WT allele and the nestin-cre transgene [16], which caused these non- Mendelian, yet consistently observed ratios. Consistent with the theory of linkage, both N-myc and the nestin-cre transgene have been mapped on mouse chromosome 12, but quite some distance apart (Roussel M. and Zindy, F., unpublished observation). In our current DKO studies, when we did an equivalent breeding (bred N-myc flox/flox; c-myc flox/flox (doubly homozygously floxed) mice with N-myc flox/WT; c-myc