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

TC 敲入：中脑边缘多巴胺分子图谱

• 批准号: 7321114
• 负责人: Cristina Backman
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON DRUG ABUSE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7321114
• 关键词: TC; Knock; Molecular; Map; Mesolimbic

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.

以前的工作： 工程细菌人造染色体（BAC）以及有效产生BAC转基因小鼠的方法的最新发展已简化了体内方法的设计，用于分析大脑中基因表达和功能。已经产生了含有外源的150 kb DNA序列的BAC转基因小鼠，该序列含有Nurr1启动子，然后直接产生了Tau绿（TG）基因。当注射到卵母细胞中时，外源性DNA在基因组中随机整合。由于BAC的承载能力为几百千碱基，因此我们能够插入150 kb的DNA片段重叠的Nurr1基因（与其他仅允许10-15 kb插入物的载体相比）。 在这些转基因小鼠中，tau绿色表达仅在嗅球中表达，并且在已知表达Nurr1的其他大脑区域不存在。该数据表明，本研究中使用的BAC（包含Nurr1启动子）缺乏在嗅球以外的其他大脑区域驱动TG表达所需的调节元素。我们目前正在通过同源重组开发其他转基因小鼠，以绘制/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，其中必须使用Nurr1/中脑多巴胺能系统的映射/可视化（见下文） 当前更新： 胚胎干细胞是通过与酪氨酸羟化酶（Th）基因的起始密码子之后引入tau-cyan（敲入）表达的cDNA编码的cDNA编码的构造来靶向的。在这些干细胞中，tau-cyan表达将由激活TH启动子的细胞中的任何元素驱动。结果，所有TH阳性细胞及其末端也将是青色阳性。这种独特的干细胞系将在各种研究中使用： 1）将生成TC转基因小鼠。这些转基因小鼠对解剖学/功能研究将很有用，其中希望对TH系统进行映射/可视化研究。激光捕获显微解剖（LCM），荧光活化的细胞分选（FACS）和贴片钳研究将通过使用这些小鼠来简化，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，这些小鼠将与含有靶向突变的小鼠进行反向跨腹膜中脑多巴胺细胞中的靶向突变（见表1），以进一步促进对中溶胶多巴胺能体系的分子和电生理研究。