To introduce restricted DNA recombination events into catecholaminergic neurons using the Cre/loxP technology, we generated transgenic mice carrying the Cre recombinase gene driven by a 9 kb rat tyrosine hydroxylase (TH) promoter. Immunohistochemistry performed on transgenic mouse brain sections revealed a high number of cells expressing Cre in areas where TH is normally expressed, including the olfactory bulb, hypothalamic and midbrain dopaminergic neurons, and the locus coeruleus. Double immunohistochemistry and immunofluorescence indicated that colocalization of TH and Cre is greater than 80%. Cre expression was also found in TH-positive amacrine neurons of the retina, chromaffin cells of the adrenal medulla, and sympathetic ganglia. We crossbred TH-Cre mice with the floxed reporter strain Z/AP and observed efficient Cre-mediated recombination in all areas expressing TH, indicating that transgenic Cre is functional. Therefore, we have generated a valuable transgenic mouse strain to induce specific mutations of "floxed" genes in catecholaminergic neurons.
The pro-opiomelanocortin (POMC) gene is expressed in a subset of hypothalamic and hindbrain neurons and in pituitary melanotrophs and corticotrophs. POMC neurons release the potent opioid beta-endorphin and several active melanocortins that control homeostasis and feeding behavior. POMC gene expression in the CNS is believed to be controlled by distinct cis-acting regulatory sequences. To analyze the transcriptional regulation of POMC in neuronal and endocrine cells, we produced transgenic mice carrying POMC27*, a transgene containing the entire 6 kb of the POMC transcriptional unit together with 13 kb of 5' flanking regions and 8 kb of 3' flanking regions. POMC27* was tagged with a heterologous 30 bp oligonucleotide in the third exon. In situ hybridization studies showed an accurate cell-specific pattern of expression of POMC27* in the arcuate nucleus and the pituitary. Hypothalamic mRNA-positive neurons colocalized entirely with beta-endorphin immunoreactivity. No ectopic transgenic expression was detected in the brain. Deletional analyses demonstrated that neuron-specific expression of POMC transgenes required distal 5' sequences localized upstream of the pituitary-responsive proximal cis-acting elements that were identified previously. POMC27* exhibited a spatial and temporal pattern of expression throughout development that exactly paralleled endogenous POMC. RNase protection assays revealed that POMC27* expression mimicked that of POMC in different areas of the CNS and most peripheral organs with no detectable ectopic expression. Hormonal regulation of POMC27* and POMC was identical in the hypothalamus and pituitary. These results show that distal 5' sequences of the POMC gene located between -13 and -2 kb target expression into the CNS of transgenic mice in a precise neuron-specific, developmentally and hormonally regulated manner.
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