Objectives: The purpose of this study is to generate BAC transgenic mice expressing human AVPR1A and to validate these animals, followed by further biochemical and behavioral analyses.
Methods: We have generated two lines with human AVPR1A by injecting BAC DNA into oocytes harvested from C57BL/6xC3H F1 hybrid mice and subsequent backcrossing of the BAC founders to wildtype C57BL6. Integration of the BAC was tested by PCR genotyping using primers directed at the non-conserved 5’UTR of gene. mRNA expression of human and murine AVPR1A was measured by quantitative RT-PCR on whole brain samples and mRNA distribution of human AVPR1A was mapped by in situ hybridization on fresh frozen slide-mounted sections using DIG-labeled RNA probes. Protein expression of human and mouse AVPR1A were mapped in transgenic and wildtype animals by performing autoradiographic ligand binding using AVPR1A selective 125I ligands.
Results: Quantitative PCR showed that whole brain levels of mouse and human AVPR1A mRNA are similar in BAC transgenic mice. In control animals, as expected, ligand binding was observed in previously documented areas including the lateral septum, bed nucleus of stria terminalis and medial amygdala. In the human BAC transgenic mice, ligand binding was more intense and widely distributed to areas in which primate AVPR1A is highly expressed such as the intralaminar nucleus of the thalamus, striatum and regions of the brain stem and spinal cord. In situ hybridization studies for localization of mRNA appear to correlate to the findings of ligand binding studies.
Conclusions: Using BAC transgenesis we generated a mouse model that robustly expresses human AVPR1A and is being used in further matings to create a fully humanized AVPR1A mouse model. Most importantly, transgenic mice show a similar expression pattern as found in humans and primates, including expression in many regions distinct from the endogenous murine receptor, indicating that human receptors are regulated by cis acting elements. Given that that differential expression patterns of AVPR1A have been suggested as important determinants of behavioral differences between species, mice expressing the human receptor may be informative of human AVPR1A signaling and circuitry with possible therapeutic relevance to ASDs.