Development of experimental models by genetic manipulation in mice has proven to be very useful in determining the significance of particular genes in the development of or susceptibility to hypertension. Advances in molecular genetics, transgenic mouse technology, and physiological measurements in mice provided an opportunity to go a step further and develop models to analyze the physiological significance of specific gene variants potentially causing hypertension. In this report, we describe the development of a human angiotensinogen transgenic mouse model generated by targeting the human angiotensinogen gene upstream of the mouse HPRT locus by homologous recombination. The main benefit of this transgenic mouse model is that the human angiotensinogen gene is inserted into the mouse genome as a single copy at a predefined locus and in a specific orientation-a process that can be repeated utilizing other variants of this gene. We establish the validity of this approach by showing that the hAGT hprt mice have normal tissue-and cell-specific expression of the human angiotensinogen gene and normally produce and process the hAGT protein at physiological levels.
Angiotensinogen (AGT) was the first gene to be genetically linked to hypertension in humans. Analysis of the gene sequence identified a number of polymorphisms, several of which were reported associated with increased blood pressure (BP) or other cardiovascular diseases. One haplotype of the human AGT (hAGT) gene consisting of an allele at the -6 (A vs. G) position in the promoter and the sequence encoding amino acid 235 (Thr vs. Met) attracted the most attention and has been the subject of numerous association studies. In this report, we addressed the physiological relevance of alleles at these two positions using an experimental mouse model system. Transgenic mice were generated by targeting each haplotype [-6G/235Met (GM) and -6A/235Thr (AT)] as a single copy transgene to the mouse hypoxanthine phosphoribosyl transferase locus, allowing direct comparison of the two transgenes in vivo. Our results indicate that both transgenes exhibit the same transcriptional activity and produce similar levels of hAGT protein in the plasma of the transgenic mice. BP analysis was performed in double transgenic mice generated by breeding each hAGT line to mice expressing a human renin gene. A small but significant increase in BP and relative heart weight was demonstrated by mice carrying the GM haplotype. Moreover, compensatory downregulation of endogenous renin expression was more pronounced in mice containing the GM variant. Our findings suggest that the AT and GM haplotypes of the hAGT gene have no effect on gene expression, but may affect the cardiovascular system and the regulation of BP differently.
With the advances in mouse molecular genetics and physiology during the last decade, the mouse has become the animal model of choice for studying the genetic basis of many diseases. Terms such as "transgenic" and "knockout" have become part of a colloquial language used in most research laboratories that are investigating human diseases. These terms refer to the two most commonly used methods for analyzing the function of a gene in vivo: overexpression (transgenic mouse) and deletion (knockout mouse). Both methods have proved to be extremely useful in establishing the importance of specific genes in genetic disorders, such as hypertension. The choice of genes being investigated in relationship to hypertension was governed by the knowledge of systems regulating vascular and renal physiology. Thus, it is not surprising that most of the focus was given to the renin-angiotensin system (RAS). Apart from the RAS, other systems known to regulate vascular tone and/or electrolyte and fluid homeostasis have also been analyzed using transgenic and knockout approaches. This review briefly summarizes some of the mouse models relevant to renal mechanisms of hypertension and then discusses the future of genetic manipulation in mice for studying the genetics of hypertension.
P13 Previous analysis of transgenic mice carrying a genomic construct encoding the human angiotensinogen (HAGT) gene indicated that the 13.7 Kb sequence harbored all cis elements required for the appropriate tissue and cell expression of HAGT in vivo . In addition, analysis of independently derived HAGT transgenic lines suggested the presence of a locus control element (LCR) in the HAGT gene since the expression levels were proportional to transgene copy number. In attempt to identify cis elements responsible for the regulation of HAGT expression in vivo we created several new transgenic mouse lines carrying either 1.5 Kb of the HAGT promoter or 1.5 kb of the promoter along with the first exon and intron of the HAGT gene fused to the LacZ reporter gene. Reporter gene expression did not correlate with transgene copy number suggesting the absence of a putative LCR element in the 1.5 Kb region proximal to the HAGT transcription start site. LacZ expression was most prominent in liver and adrenal gland, with additional expression in heart, testis, brain, and cerebellum. Most other tissues (including kidney) had little to no expression of the reporter gene. Our results indicate that the regions proximal to the HAGT transcription start site contain cis -acting elements important for appropriate HAGT expression in the liver and some, but not all, extra-hepatic tissues which should express HAGT. Further dissection of the genomic HAGT sequence will be required to identify all elements involved in proper in vivo regulation of the HAGT gene.
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