Figure 1. Targeted disruption of the murine Pde3b gene.

(A) Structure of the approximately 13-kb SalIPDE3B WT genomic fragment containing 5′-untranslated region and exon 1. (B) WT and disrupted KO gene fragments near exon 1. (C) Pde3b targeting vector. S, SalI; X, XbaI; B, BstXI; N, NotI; H, HindIII. (D) Southern blot of WT, HE, and KO genomic DNA, digested with BstXI and hybridized to ³²P-labeled probe 0.4 (see A and C). (E) PCR amplification of WT, HE, and KO genomic DNA using the specific primers A, E, and R indicated in A and B (A and E for lane 1; A and R for lane 2). (F) RT-PCR amplification of mRNA from WT, HE, and KO livers with primers described in Methods targeted for PDE3A (lane 1), PDE3B (lane 2), and Neo^r (lane 3). M, molecular weight marker. (G) Quantitation of cyclophilin A (Cyc), PDE3A, and PDE3B mRNAs in WT, HE, and KO mouse livers by real-time RT-PCR. Data were normalized to the quantity of WT cyclophilin A mRNA, taken as 100 AU. Values represent mean ± SEM (n = 4 per genotype in triplicate assays). Data were similar in 2 other groups of WT and KO mice and 1 group of HE mice. **P < 0.01. (H and I) Northern blot of WT and KO mRNAs, using probe B for mPDE3B (fat pads and liver; H) or probe A for mPDE3A (heart; I) as described in Methods. M, male; F, female; W, WT mRNA; K, KO mRNA.