TC Knock-in:Molecular Map-Mesolimbic Dopaminergic System

以前的工作： 近年来，设计细菌人工染色体(BAC)和高效生产BAC转基因小鼠的方法的发展，简化了体内基因表达和脑功能分析方法的设计。BAC转基因小鼠携带外源150kb的DNA序列，该DNA序列包含Nurr1启动子和Tau green(TG)基因。当注射到卵母细胞中时，外源DNA随机整合到基因组中。由于BAC的载量为几百个碱基，我们能够插入与Nurr1基因重叠的150kb的DNA片段(与其他载体相比，只允许10-15kb的插入)。 在这些转基因小鼠中，Tau绿的表达只在嗅球中表达，而在其他已知表达Nurr1的大脑区域中没有表达。这一数据表明，本研究中使用的BAC(包含Nurr1启动子)缺乏一些在嗅球以外的大脑区域驱动TG表达所必需的调节元件。我们目前正在开发更多的BAC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学/功能研究，其中需要对Nurr1/中脑多巴胺能系统进行映射/可视化(见下文)。 当前更新： 胚胎干细胞正通过同源重组成为靶点，在酪氨酸羟基酶(TH)基因的终止密码子之后引入编码Tau-Cyan(敲入)表达的cDNAs。在这些干细胞中，Tau-Cyan的表达将由细胞中任何激活TH启动子的元素驱动。因此，所有TH阳性细胞及其终末也将呈青色阳性。这种独特的干细胞株将用于各种研究： 1)建立TC转基因小鼠。这些转基因小鼠将用于解剖/功能研究，其中TH系统的图谱/可视化研究是可取的。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，因为显示TH-Cyan阳性细胞不需要免疫染色或标记。在我们的实验室中，这些小鼠将与含有腹侧中脑多巴胺细胞靶向突变的小鼠进行回交(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 BAC transgenic mice, 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 stop 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.