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

以前的工作： 最近开发的工程细菌人工染色体（BAC）的方法，并为BAC转基因小鼠的有效生产，简化了体内方法的设计，在大脑中的基因表达和功能的分析。已经产生了携带外源性150 kb DNA序列的BAC转基因小鼠，所述外源性150 kb DNA序列含有Nurr 1启动子，紧接着是Tau绿色（TG）基因。外源DNA注入卵母细胞后，随机整合到基因组中。由于BAC的携带能力是几百个内切酶，我们能够插入与Nurr 1基因重叠的150 Kb DNA片段（与仅允许10-15 kb插入的其它携带载体相比）。 在这些转基因小鼠中，Tau绿色表达仅在嗅球中表达，而在已知表达Nurr 1的其他脑区中不表达。这些数据表明，在这项研究中使用的BAC（含有Nurr 1启动子）缺乏一些必要的调控元件驱动TG的表达在大脑区域以外的嗅球。我们目前正在通过同源重组开发更多的转基因小鼠，用于绘制/可视化中脑多巴胺能系统。这些小鼠将在我们的实验室中用于进行几项解剖/功能研究，其中Nurr 1/中脑多巴胺能系统的映射/可视化是必要的（见下文） 当前更新： 胚胎干细胞通过与在酪氨酸羟化酶（TH）基因的起始密码子之后引入编码Tau-Cyan（敲入）表达的cDNA的构建体的同源重组而被靶向。在这些干细胞中，Tau-Cyan表达将由细胞中存在的激活TH启动子的任何元件驱动。因此，所有TH阳性细胞及其末端也将是青色阳性的。这种独特的干细胞系将用于各种研究： 1)将产生TC转基因小鼠。这些转基因小鼠将是有用的解剖/功能研究，其中映射/可视化研究TH系统是可取的。使用这些小鼠将简化激光捕获显微切割（LCM）、荧光激活细胞分选（FACS）和膜片钳研究，因为不需要免疫染色或标记来可视化TH-Cyan阳性细胞。在我们的实验室中，这些小鼠将与腹侧中脑多巴胺细胞中含有靶向突变的小鼠回交（见表1），以进一步促进用特定突变修饰的中脑边缘多巴胺能系统的分子和电生理学研究。

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.