TC Knock-in: A Molecular Map of the Mesolimbic Dopaminer

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PREVIOUS WORK: The recent development of methods for engineering bacterial artificial chromosomes (BACs), and for the efficient production of BAC transgenic mice, has simplified the design of in vivo approaches for the analysis of gene expression and function in the brain. BAC transgenic mice carrying an exogenous 150 kb DNA sequence containing the Nurr1 promoter directly followed by the Tau green (TG) gene have been generated. The exogenous DNA, was randomly integrated in the genome when injected into oocytes. As the carrying capacity of BACs is several hundred kilobases, we were able to insert a 150 Kb DNA fragment overlapping the Nurr1 gene (as compared to other carrying vectors allowing only 10-15 kb inserts). In these transgenic mice, Tau green expression was expressed only in the olfactory bulb, and absent in other brain areas known to express Nurr1. This data suggests that the BAC (containing the Nurr1 promoter) used in this study is lacking some of the regulatory elements necessary for driving TG expression in brain regions other than the olfactory bulb. We are currently developing additional transgenic mice by homologous recombination, for mapping/visualizing the midbrain dopaminergic system. These mice will be used in our laboratory to conduct several anatomical/functional studies in which mapping/visualization of the Nurr1/midbrain dopaminergic system is necessary (see below) CURRENT UPDATE: Embryonic stem cells are being targeted by homologous recombination with a construct that introduces cDNA encoding for Tau-Cyan (knock-in) expression after the start codon of the tyrosine hydroxylase (TH) gene. In these stem cells, Tau-Cyan expression will be driven by any elements present in the cell that activate the TH promoter. As a result all TH positive cells and their terminals will also be Cyan positive. This unique stem cell line will be used in various studies: 1) TC transgenic mice will be generated. These transgenic mice will be useful for anatomical/functional studies in which mapping/visualization studies of the TH system are desirable. Laser capture microdissection (LCM), fluorescence activated cell sorting (FACS) and patch-clamp studies will be simplified by using these mice, since no immunostaining or labeling will be required to visualize TH-Cyan positive cells. In our laboratory, these mice will be backcrossed with mice containing targeted mutations in dopamine cells of the ventral mesencephalon (see table 1) to further facilitate molecular and electrophysiological studies of the mesolimbic dopaminergic system modified with specific mutations.