Isolation and immortalization of MIP-GFP neurons from the hypothalamus

The mouse insulin I promoter (MIP) construct was developed to eliminate the promoter activity detected with the rat insulin II promoter in specific hypothalamic neurons that may have unintended effects on glucose and energy homeostasis in transgenic models. Thus, the specificity of this novel construct must be validated prior to the widespread availability of derived Cre models. Although limited validation efforts have indicated a lack of MIP activity within neuronal tissue, the global immunohistochemical methodology used may not be specific enough to rule out the possibility of specific populations of neurons with MIP activity. To investigate possible MIP activity within the hypothalamus, primary hypothalamic isolates from MIP-green fluorescent protein reporter mice were analyzed after fluorescent-activated cell sorting. Primary hypothalamic neurons isolated from the MIP-green fluorescent protein mice were immortalized. Characterization detected the presence of hypothalamic neuropeptide Y (NPY) and agouti-related peptide, involved in the control of energy homeostasis, as well as confirmed insulin responsiveness in the cell lines. Moreover, because insulin was demonstrated to differentially regulate NPY expression within these MIP neurons, the promoter construct may be active in multiple hypothalamic NPY/agouti-related peptide subpopulations with unique physiological functions. MIP transgenic animals may therefore face similar limitations seen previously with rat insulin II promoter-based models.