Caron et al. 10.1073/pnas.0611365104. |
SI Materials and Methods
Construction of Gene Targeting Vector.
To generate the targeting vector, a 129S6/SvEv genomic library was screened for phage clones containing the AM gene. By using convenient restriction sites within the genomic clone, 5' and 3' regions of homology were subcloned into the multiple cloning site of a gene targeting vector (osdupdel) which contains a PGK-neomycin cassette and an a HSV-thymidine kinase cassette. The final targeting vector was linearized with NotI before electroporation into ES cells.Generation of Targeted ES Cells and Mice.
Standard gene-targeting methods were used to generate ES cells and mice with a targeted duplication of the AM gene (1). Briefly, 129S6/SvEv-TC-1 ES cells were electroporated with the linearized targeting vector shown in Fig. 1A. After applying positive (G418) and negative (ganciclovir) selection, positive ES cell clones were identified by Southern blot and/or PCR. The frequency of homologous recombination was 3% for the AM gene duplication. Male chimeric mice that transmitted the targeted allele were bred to 129S6/SvEv females to establish isogenic lines. Genotyping for the duplicated AM gene was performed by Southern blot as described in Fig. 1.Measurement of AM Levels.
AM gene expression was assayed by quantitative RT-PCR (2). Tissue levels of AM peptide were measured by using a RIA (3). Plasma levels of AM peptide were not measured because of small sample volumes.Measurement of BP.
BPs were measured on unanesthetized mice by a computerized tail-cuff system, as described (4). Either male or nonpregnant female mice were implanted with BP transducers (Data Sciences International, St. Paul, MN) under anesthesia with 1.5-2.0% isoflurane/oxygen gas. A ventral midline neck incision was made, the left common carotid artery was isolated and retracted, and a small incision was made. The pressure-sensing catheter was carefully inserted into the left carotid artery and was advanced 10 mm so that its tip resided just inside the thoracic aorta. The catheter was then secured by ligatures, and the transmitter body was tunneled s.c. to a small pouch along the right ventral flank. The neck incision was closed with 6-0 silk, and the mice were kept warm until they had fully recovered from anesthesia. After 6 days of recovery in home cages, transmitters were magnetically activated, and baseline mean arterial BP was recorded continuously for 5 days (15 measurements/hour). The data were collected, stored, and analyzed by using Dataquest A.R.T. software (Data Sciences). For the pregnancy studies, male mice were introduced into the cages and the date of the vaginal plug was recorded as e0.5. Six pregnancies were monitored in four mice.Lipopolysaccharide-Induced Septic Shock.
Septic shock was induced in wild-type and AM 1 copy mice by i.p. injection of 700 mg/kg D-galactosamine and 40 mg/kg lipopolysaccharide E. Coli O111:B4. Changes in BP after treatment were monitored by telemetry, as described above.Hypertension and Cardiac Hypertrophy Induced by a Genetically Clamped Renin Transgene.
We have previously described the generation and characterization of a mouse line in which a renin transgene (RenTgMK), targeted as a single copy between the Apoa1 and Apoc3 genes, is expressed by the liver and results in hypertension, renal damage, and cardiac hypertrophy (5-7). To genetically induce hypertension and cardiac hypertrophy in the AM one-copy mice, heterozygous RenTgMK mice were crossed to heterozygous AM one-copy mice. All mice were maintained on an isogenic SvEv129/S6 genetic background and were 6-8 months of age at the time they were killed.Histology.
Heart and kidneys were dissected, weighed, and fixed in 4% paraformaldehyde overnight. The samples were then dehydrated and embedded in paraffin wax, and 5-mm sections were stained with Masson's Trichrome reagent. For morphometry studies, cross-ectional areas of five myocytes from three different sections of four to six mice were measured by using Image J software (NIH version 2). Scoring of renal damage was performed in a blinded fashion. A scale of 1-4 was established, with 1 being unaffected, and 4 being severely affected based on the following parameters: glomerular sclerosis, vascular fibrosis, interstitial fibrosis, proteinaceous casts. Scoring was performed on two sections from four mice for each gender and genotype.Experimental Animals.
Experimental animals were 6-8 months of age and maintained on an isogenic 129S6/SvEv-TC-1 background. Control animals were wild-type, age- and gender-matched littermates. All experiments were approved by the Institutional Animal Care and Use Committee of The University of North Carolina, Chapel Hill, NC.Statistics.
Statistical analyses for multiple comparisons were performed with one-way ANOVA using JMP Software, SAS Institute. In all figures, error bars represent standard errors of the mean. Differences were considered significant with a P < 0.05.1. Smithies O, Kim HS (1994) Proc Natl Acad Sci USA 91:3612-3615.
2. Caron KM, Smithies O (2001) Proc Natl Acad Sci USA 98:615-619.
3. Nishikimi T, Horio T, Sasaki T, Yoshihara F, Takishita S, Miyata A, Matsuo H, Kangawa K (1997) Hypertension 30:1369-1375.
4. Krege JH, Hodgin JB, Hagaman JR, Smithies O (1995) Hypertension 25:1111-1115.
5. Caron KM, James LR, Lee G, Kim HS, Smithies O (2005) Physiol Genomics 20:203-209.
6. Caron KM, James LR, Kim HS, Knowles J, Uhlir R, Mao L, Hagaman JR, Cascio W, Rockman H, Smithies O (2004) Proc Natl Acad Sci USA 101:3106-3111.
7. Caron KM, James LR, Kim HS, Morham SG, Sequeira Lopez ML, Gomez RA, Reudelhuber TL, Smithies O (2002) Proc Natl Acad Sci USA 99:8248-8252.