This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The objectives of producing these transgenic rat models are to study the regulation of mineralocorticoid binding ligands in different tissues. To this end rats expressing the siRNA for 11?-hydroxysteroid dehydrogenase 2 (11-HSD2) under control of the U6 promoter and those over-expressing aldosterone synthase driven by a neuron-selective synapsin 1 promoter (ASs1) or myosin heavy chain promoter to target the heart have been produced. One-cell rat embryos were injected under the zona pellucida with a lentivirus. On the first try, despite the presence of the 11-HSD2 siRNA transgene in 19% of the progeny, the phenotype of the F2 and F3 generations, as assessed by steroid metabolism profile and western blots of kidney biopsy samples, showed no decrease in enzyme expression. The second try with this transcript has produced one founder animal with a significant decrease in 11-HSD expression in the kidney. She is expected to whelp next week. We obtained 13 founder ASs1 rats that had significantly higher systolic blood pressure than their wt siblings on a normal salt diet. Five founder F1 families were selected on the basis of the number of real time RT-PCR positive progeny in a litter and their systolic blood pressure. Plasma steroids and inflammatory cytokines are being determined for the F2 heterozygotes from the 5 different families. The brains from these F2 ASs1 are being assessed by immunohistochemistry for AS expression and hearts and kidneys are being studied for markers of hypertrophy and inflammation. Those F3 individuals that appear by real time RT-PCR to be homozygotes are being bred with wt rats to ascertain their homozygosity. Once identified, they will be bred and the homozygote phenotype further characterized. We have become fairly consistent with the technique of rat embryo cryopreservation and successful production of adults, so will be cryopreserving candidate strains.
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