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S.H. p35C/Cp39C/C double-null mutants are perinatal lethal. We show here that the p35C/Cp39C/C mutants exhibit phenotypes identical to those of the Cdk5-null mutant mice. Other compound-mutant mice with intermediate phenotypes allow us to determine the distinct and redundant functions between p35 and p39. Our data strongly suggest that p35 and p39 are essential for Cdk5 activity during the development of the nervous system. Thus, p35 and p39 are likely to be the principal, if not the only, activators of Cdk5. during development of the nervous system, we created mice with a targeted deletion in the p39 locus. In addition, we generated p35/p39 compound mutants to test the hypothesis that p35 and p39 are the essential two activators for Cdk5 function and to elucidate further the roles of p35 and p39 during neurodevelopment. In this study, we show that p39-deficient mice do not exhibit detectable abnormalities in neuronal positioning in the nervous system. However, p35C/Cp39C/Cdouble-null mutants display phenotypes that are identical to those of Cdk5C/C mice in all neuronal compartments analyzed, indicating that p35 and p39 are indeed essential activators of Cdk5. Comparative analysis of gene dosage effects of p35 and p39 in the single and compound mutants reveals that p35 and p39 play distinct roles in the regulation of Cdk5 activity and have differential compensatory capabilities in particular brain compartments. Moreover, we have novel data to suggest that the role of Cdk5 in neurodevelopment extends beyond the scope established previously. MATERIALS AND METHODS The murine homolog of p39 was isolated and characterized (Nilden et al., 1998). Corresponding genomic clones were obtained by screening of a 129/Sv mouse genomic library (Stratagene, La Jolla, CA) with a PCR product containing the entire p39 open reading frame. To construct the targeting vector (see Fig. ?Fig.11cassette in the reverse orientation replaced 1 kb of the 1.1 kb open reading frame of the p39 locus. denote the denote the regions amplified by PCR genotyping.band of 650 bp. The presence of both bands denotes a heterozygous animal. Compound-mutant mice were generated via a two- or three-stage cross. p35C/C and p39C/C mice were mated to produce p35+/Cp39+/CF1 progeny. F1 progeny were self-crossed to obtain compound-mutant mice, 6.25% of which were expected to be double knockouts. An additional cross was implemented with F2 progeny of p35+/Cp39C/Cand p35C/Cp39+/Cgenotypes to produce litters of which 25% of pups will be double knockouts. p35 genotyping was performed using PCR as described previously (Kwon and Tsai, 1998). The p39 genotype was determined by Southern blot (see above) and by PCR analysis of genomic DNA prepared from tails. Two sets of PCRs allowed for the determination of genotype. Reaction 1 amplifies the region of 350 bp present only in nonrecombinant genomic DNA TCS ERK 11e (VX-11e) at the p39 gene locus (primer p39 up, AGC TTC GGC GGC TCA GAG CAG CC; primer p39 down, AGC ACG GAC GGC CGT TTG AGC). Reaction 2 amplifies the region of 650 bp specific TCS ERK 11e (VX-11e) to the genomic deletion (primers p39 up and neo, GAT ATT GCT GAA GAG CTT GGC). Heads and bodies of E18. 5 mice were dehydrated and embedded in paraffin. Serial coronal, parasagittal, and transverse tissue sections were cut at 6C7 m and stained with hematoxylin and eosin for histopathological analysis. Two to three specimens of each genotype were analyzed, and littermates were used for comparison whenever possible. Calbindin D-28-K (Sigma, St. Louis, MO; 1:3000) and calretinin (SWANT, Bellinzona, Switzerland; 1:1000) immunoperoxidase staining was performed on paraformaldehyde-fixed tissue according to established protocols using the avidinCbiotinCperoxidase complex technique (ABC; Vector Laboratories, Burlingame, CA). SMI34 (Sternberger Monoclonals, Lutherville, MD; 1:10,000) immunoperoxidase staining was performed on Bouin’s fixed tissue. Whole adult and postnatal day 0 (P0) mouse brains were lysed in 500 l of radioimmunoprecipitation (RIPA) buffer (150 mm NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mmTris, pH 8) or E1A lysis buffer (50 mmHEPES, pH 7.0, 0.1% NP-40, 250 mm NaCl, and TCS ERK 11e (VX-11e) 5 mm EDTA, pH 8) with protease and phosphatase inhibitors (1 mm Na3VO4, 50 mm NaF, 1 mm PMSF, 1 TCS ERK 11e (VX-11e) g/ml aprotinin, 10 g/ml leupeptin, 1 nm okadaic acid, 5 m pyrophosphate, 1 mm DTT, and 50 m -glycerophosphate) in a homogenizing Dounce and then spun at 13,000 rpm for 15 min at 4C. The supernatant was collected, and the amount of protein was determined by the Bradford procedure using the Bio-Rad protein assay solution (Bio-Rad, Hercules, TCS ERK 11e (VX-11e) CA). Fifteen DDIT1 micrograms of total proteins were loaded per lane on an 8% acrylamide/bis-acrylamide (30%/0.8%) gel and separated by SDS-PAGE. Proteins were then transferred to Immobilon-P transfer membrane (Millipore, Bedford, MA). Blots were then subjected to.