Am J Pathol. 2008 July; 173(1): 42–56.

Genetic and Biometric Characteristics of Dox-Treated Transgenic CCSP/FasL Mice at E19 Age E19 Genotype CCSP+/FasL+ CCSP+/FasL− Fraction of living offspring 24/51 (47%) 27/51 (53%) Body weight (g) 1.11 ± 0.11 (24) 1.11 ± 0.11 (27) Lung weight (g) 0.023 ± 0.006 (17) 0.026 ± 0.006 (21) Lung weight/body weight (%) 2.16 ± 0.54 (17) 2.42 ± 0.01 (21)* Lung weights at E19 reflect wet lung weights. Values represent mean ± SD. *P < 0.05 versus CCSP+/FasL+ littermates (Student’s t-test). Am J Pathol. 2008 July; 173(1): 42–56.

Am J Pathol. 2008 July; 173(1): 68–76.

Generation of Mice KO (Smad3ex8/ex8) mice were generated by targeted disruption of the Smad3 gene by homologous recombination15 and are a mixed background of C57BL/6nCr, Tac:N:NIHS-BCFBR, and 129S6/SvEv-ATM<tm1awb>, which we have used previously.9 Mice heterozygous for the targeted disruption were intercrossed to produce homozygous offspring and genotyping was done by polymerase chain reaction of tail DNA.

Am J Physiol Heart Circ Physiol 280: H368-H376, 2001;

Generation of transgenic mice. Standard procedures were used to generate transgenic mice (16). Mice were produced at the Transgenic/Knockout Animal Facility Core at the National Institute of Environmental Health Sciences Center for Molecular and Cellular Toxicology at Wayne State University (Detroit, MI). The proximal hBNP promoter (408 to +100) was cloned upstream from a luciferase reporter gene in a pUC18 plasmid (20). The transgene was removed from the plasmid by double digestion with Hind III and Pvu II, purified, and microinjected into zygotes from B6C3F1 mice. Viable injected zygotes were transferred into the infundibulum of the oviduct in pseudopregnant CD-1 mice. Three weeks after birth, genomic DNA was extracted from a 1.0-cm piece of tail and subjected to Southern blot for detection of the transgene. Founder mice (transgene-positive) were mated with nontransgenic littermates to test for transmission of the transgene. Positive offspring were mated to produce litters that were heterozygous or homozygous at the transgene locus. Male and female heterozygous and homozygous mice were used in these experiments.

J. Clin. Invest. 118(1): 29-39 (2007)

Generation of RAMP2 KO mice. KO mice were generated as described previously (14, 16, 41, 42). Briefly, a plasmid-targeting vector was constructed to insert loxP sites encompassing exons 2–4 of RAMP2 and the neomycin resistance gene, after which the plasmid was linearized and introduced into Bruce 4 embryonic stem cells by electroporation. Homologous recombinants were identified, and 2 independently targeted clones were injected into BALB/c blastocysts to generate chimeric mice. Male chimeras were crossbred with C57BL/6 females, and germline transmission was verified by Southern blot analysis. After obtaining heterozygotic floxed RAMP2 mice, we crossbred them with CAG-Cre mice to delete exons 2–4 of the RAMP2 gene. The deletion of RAMP2 was certified by Southern blot analysis. The Cre gene was then removed from the line by backcrossing with C57BL/6 mice. All experiments were performed in accordance with the Declaration of Helsinki and were approved by the Shinshu University Ethics Committee for Animal Experiments.

J. Clin. Invest. 118(1): 40-50 (2007)

Adrenomedullin signaling is necessary for murine lymphatic vascular developmentJ. Clin. Invest. Kimberly L. Fritz-Six, et al. 118:40 doi:10.1172/JCI33302 [Go to this article.] Figure 2Generation and characterization of conditional calcrl line. (A) Schematic diagram depicting strategy used for generation of a floxed calcrl allele by gene targeting. The top figure shows the endogenous wild-type calcrl allele. The targeting vector was designed so that loxP sites would flank the same exons that were deleted in the calcrl global knockout (36). The third line depicts the targeted calcrlFlox allele, and the fourth line depicts the calcrlLoxP allele after Cre-mediated excision. Primers used for isolation of correctly targeted ES cells and for routine genotyping are indicated by small arrows. (B) Correctly targeted ES cells were confirmed by Southern blot analysis using the probe depicted in A. (C) PCR genotyping for the calcrlFlox allele. (D) Quantitative RT-PCR was performed on RNA isolated from lungs and hearts of wild-type and homozygous calcrlFlox/Flox mice and revealed no significant differences in the expression of the calcrlFlox allele before Cre-mediated excision. (E) Schematic representation of breeding scheme used to generate mice with calcrl expression deleted specifically in endothelial cells by use of the Tie2Cre transgene. (F) Results of cross demonstrate that no viable calcrlLoxP/–Tie2Cre+ mice were found beyond E16.5. (G) Compared with calcrlLoxP/+Tie2Cre+ control littermates, the calcrlLoxP/–Tie2Cre+ mice displayed remarkable hydrops without hemorrhage, which phenocopied the global calcrl–knockout phenotype, yet often occurred substantially later, at E16.5. Original magnification, ×10.

J. Clin. Invest. 118(1): 124-132 (2007).

The MEF2D transcription factor mediates stress-dependent cardiac remodeling in miceJ. Clin. Invest. Yuri Kim, et al. 118:124 doi:10.1172/JCI33255 [Go to this article.] Figure 1Generation of mice with a conditional Mef2d mutation. (A) Schematic representation of the mouse Mef2d locus and targeting strategy. Positions of 3ι and 5ι probes used for Southern blots are shown. The positions of the PCR primers used for genotyping mutant alleles are marked with arrows (circles labeled 1, 2, and 3). C, ClaI; frt, FLP recombinase target; E, EcoRI; TAD, transactivation domain; MADS, MCM1, agamous, deficiens, serum response factor; N, NcoI; X, XhoI. (B) Southern blot analysis of Mef2d mutant alleles. Genomic DNA was digested with EcoRI and hybridized to a 5ι probe in the left panel and to a 3ι probe in the right panel. WT, wild-type allele; Mef2dneo-loxP, conditional allele; Mef2dloxP, conditional allele with the Neo cassette removed; Mef2dKO, null allele. (C) PCR genotyping to distinguish different Mef2d alleles. The positions of the primers that produce these PCR products are labeled (1, 2, and 3) and circled on A. All 3 primers were added to the PCR reactions and the PCR products were loaded into lanes 2–7. (D) Expression of wild-type and mutant Mef2d detected by RT-PCR. Mef2d mutant allele lacks exon 3, which encodes the MADS- and MEF2-specific domains. GAPDH was used as a loading control. Labels on the left side of the panel indicate exon location and direction of primers used for RT-PCR. (E) Western blot analysis to detect WT and mutant MEF2D (KO) proteins. The mutant MEF2D protein is trunca-ted due to deletion of exon 3. α-Actin protein was used as a loading control. (F) Expression level of Mef2 detected by quantitative PCR. Error bars indicate ±SEM. u.d., undetectable.

J. Clin. Invest. 118(1): 124-132 (2007).

The MEF2D transcription factor mediates stress-dependent cardiac remodeling in miceJ. Clin. Invest. Yuri Kim, et al. 118:124 doi:10.1172/JCI33255 [Go to this article.] Figure 2Blunted hypertrophy of Mef2d mutant mice following TAC. (A) HW/TL ratios (±SEM) of WT and Mef2d mutant mice were determined 21 days after TAC. (B) Hearts from WT and Mef2d mutant mice subjected to either a sham operation or pressure overload (TAC) are shown at the top. Histological sections stained with Masson’s trichrome are shown on the bottom. Masson’s trichrome staining of WT and Mef2d mutant hearts indicates lack of fibrosis in Mef2d–/– hearts in response to pressure overload by TAC. Scale bars: 1 mm (middle panel); 40 μm (bottom panel). (C) Mean cross-sectional area of cardiomyocytes (±SEM) in WT and Mef2d mutant mice was measured 21 days after TAC. (D) Using morphometric analysis of picrosirius red stained heart sections, the amount of myocardial fibrosis was assessed. Fibrosis was apparent in the hearts of WT mice following TAC. In contrast, virtually no fibrosis was detected in the hearts of Mef2d mutant mice. Values indicate fold changes of fibrosis in each group compared with a group of sham-operated WT mice (±SEM).

Circulation. 2008;118:157-165

Figure 1. Impairment of endothelial commitment, proliferation, and invasiveness of EPCs in BM of conditional Jag-1–/– mice. A, Generation of conditional knockout (KO) mice lacking Jag-1 or DII-1 gene. Reverse-transcription polymerase chain reaction confirmed specific deletion of Jag-1 or Dll-1 mRNA expression in BM stromal cells obtained from each of the KO mice. WT indicates wild type. B, Frequency of KSLs in BM was similar in WT, Jag-1 KO, and Dll-1 KO mice (n=3 in each group). C, Fluorescence-activated cell sorting analysis for CD31 and Flt-1 (VEGFR1) or Flk-1 (VEGFR2) using BM Sca-1+/Lin– cells obtained from WT, Jag-1 KO, or Dll-1 KO mice. Frequency of Flt-1+/CD31+ or Flk-1+/CD31+ cells, which are EPC-enriched populations, in Sca-1+/Lin– cells was drastically lower in Jag-1 KO mice but not in Dll-1 KO mice compared with WT mice (n=3 in each group). D, EPC colony-forming assay was performed by incubation of BM-KSLs from WT, Jag-1 KO, or Dll-1 KO mice in methyl cellulose–containing medium, supplemented by several cytokines as the driving force for endothelial differentiation. Left, Representative EPC colony clusters with a spindlelike morphology; Right, significant inhibition of EPC colony-forming capacity in Jag-1 KO mice but not Dll-1 KO mice compared with WT mice. CFU indicates colony-forming unit. **P<0.01 (n=3 in each group). E, BrdU proliferation assay was performed with BM Sca-1+/Lin– cells of WT, Jag-1 KO, or Dll-1 KO mice 14 days after induction of hindlimb ischemia. Frequency of BrdU+/Sca-1+ cells in BM-Lin– cells was significantly lower in Jag-1 KO mice but not Dll-1 KO mice compared with WT mice. **P<0.01 (n=5 per group). F, Frequency of TUNEL-positive cells in BM Sca-1+/Lin– cells 3 days after hindlimb ischemia was significantly greater in Jag-1 KO mice than in Dll-1 KO and WT mice. *P<0.05 (n=3 in each group). G, In vitro invasiveness assay with BM Sca-1+/Lin– cells from WT, Jag-1 KO, or Dll-1 KO mice 3 days after hindlimb ischemia. Number of cells invading into methyl cellulose was significantly lower in Jag-1 KO mice but not Dll-1 KO mice compared with WT in the presence of stromal cell–derived factor-1 (SDF-1), although invasiveness activity was similar in all groups in the absence of stromal cell–derived factor-1. HPF indicates high-power field. **P<0.01 (n=4 per group).

Circulation. 2008;118:157-165

Figure 5. Promotion of in vivo neovascularization by transplantation of putative EPCs stimulated by Jag-1–mediated signals. A, Representative laser Doppler perfusion imaging findings in nude mice receiving PBS (no cells) or BM-Lin– cells cocultured with empty-vector– or specific Notch ligand (Jag-1, Dll-1)–transfected 3T3 stromal cells at days 0 and 14 (upper panel). Hindlimb perfusion recovery was significantly enhanced in the Jag-1 group compared with the Dll-1, empty-vector, and PBS groups (n=6 per group, lower panel). *P<0.05 vs PBS; **P<0.01 vs PBS; ***P<0.001 vs PBS; P<0.05 vs vector; P<0.01 vs vector; P<0.05 vs Dll-1; P<0.01 vs Dll-1. B, Histological capillary density by isolectin B4 staining revealed augmented neovascularization in the Jag-1 group but not the Dll-1 group compared with the PBS group. *P<0.05; **P<0.01 (n=4 per group). HPF indicates high-power field. C, Histological density of putative EPCs (BM-Lin– cells obtained from GFP transgenic mice) incorporating into vasculature of ischemic tissue. The density of the incorporating EPCs identified as CD31+/GFP+ cells was significantly greater in the Jag-1 group than in the Dll-1, empty-vector, and PBS groups. Green fluorescence indicates GFP; red signal, CD31. *P<0.05; **P<0.01 (n=4 per group).

J Clin Invest. 2008 January 2; 118(1): 100–110

Figure 1 Generation of Trail-r–/– mice. (A) Strategy to target exon 2 of the Trail-r gene. (i) Functional protein domains of TRAIL-R encoded by the Trail-r WT allele (ii). SP, signal peptide; CRD, cysteine-rich domain; TM, transmembrane domain; DD, death domain. Locations of the external (ext) 3′ and 5′ and the internal (int) probes and of genotyping primers mTR-a (a), mTR-b (b), and mTR-d (d) are indicated. (iii) Targeting construct. Open triangle, FRT sites; gray triangles, loxP sites; tkneo, selection cassette; DTA, diphtheria-toxin A-cassette. (iv) Null allele. (B) Southern blot analysis of targeted G418-selected ES cell clone after correct homologous recombination. (C) Constitutive deletion of exon 2 of Trail-r in different tissues. PCR analysis of genomic DNA of tail, lymph node, kidney, lung, heart, and brain of Trail-r–/–, Trail-r+/–, and Trail-r+/+ mice using primers a, b, and d is shown. The primers a and b amplified a 300-bp fragment of the Trail-r WT allele, and primers a and d amplified a 235-bp fragment of the Trail-r–null allele. J Clin Invest. 2008 January 2; 118(1): 100–110.

J Clin Invest. 2008 January 2; 118(1): 100–110

Generation of Trail-r–/– mice. A genomic BAC clone containing 10 kb of the Trail-r (Tnfrsf10b) gene (Figure 1A) was isolated from a genomic phage library derived from male ES cells of the mouse strain 129/Sv (Mobitec). The targeting construct for generation of conditional and constitutive Trail-r–/– mice (Figure 1A) was cloned by 2 rounds of ET cloning (RecE and RecT recombination) in E. coli, as previously described (47). The predicted polypeptide encoded by the null allele contains only the signal peptide, but did not encode the pre–ligand-binding assembly domain, the TRAIL-binding cysteine-rich domains, or the death domain (Figure 1A). The construct consisted of a 4.5-kb 5′ homology arm and a 3.1-kb 3′ homology arm, a tkneo selection cassette for positive selection by G418, and a diphtheria–toxin A (DTA) selection cassette for negative selection during homologous recombination at the 3′ end. Two loxP sites (recognized by the Cre recombinase) flanked a sequence containing exon 2 and the tkneo cassette, while the tkneo cassette alone was additionally flanked by 2 FRT sites (recognized by the Flp recombinase) (Figure 1A). ES cells of the 129/Ola strain were transfected with the linearized targeting construct (Figure 1A). We isolated G418-resistant ES cell clones and confirmed homologous recombination by restriction digest of genomic DNA followed by Southern blot analysis using external 3′ and 5′ probes (3′-ext and 5′-ext) and an internal probe (Figure 1, A and B). In a correctly recombined ES cell clone, the tkneo selection cassette was excised by transfection with a eukaryotic expression vector encoding Flp recombinase (pCAGGS-FLP-e) (48) followed by negative selection with gancyclovir, which is toxic for cells expressing thymidine kinase. Correct excision of the selection cassette in ES cells was confirmed by PCR and Southern blot analysis (data not shown). ES cells containing the conditional Trail-rflox allele were injected into blastocysts, and chimeric mice were tested for germline transmission by PCR (data not shown). To generate the Trail-rnullallele, heterozygous conditional Trail-rflox/+ animals were crossbred to mice expressing the Cre recombinase under the control of the Nestin promoter, which is not only expressed in the brain but occasionally also in the germline (our unpublished observations). Constitutive germline deletion of exon 2 of Trail-r was determined by PCR genotyping analysis of genomic DNA of tail, lymph node, kidney, lung, heart, and brain (Figure 1C). RT-PCR analysis confirmed excision of exon 2 at the mRNA level (data not shown). Backcrossed mice were selected for loss of Cre recombinase.