Generation Brain Subregion-Conditional Transgenic Mice

大脑的巨大复杂性源于数百种神经细胞类型和它们之间广泛的突触连接。最近，基因工程的发展彻底改变了脑亚区局部功能的研究，该技术理想地开关了体内特定脑亚区特定细胞类型的基因表达。例如，P1噬菌体的Cre重组酶已被证明对成人大脑有丝分裂后神经元细胞的有条件转基因操作是非常宝贵的。自2003年3月以来，我们启动了一个项目，创建各种脑亚区或细胞类型受限的条件转基因小鼠。我们今年的目标是利用携带启动子的BAC（细菌人工染色体）克隆，创建各种亚区限制性crer -重组酶转基因系，以指导特定大脑区域的表达。从文献和我们的原位杂交组织化学中，我们发现一些BAC克隆携带的基因启动子主要表达在海马CA1（即BAC7）、杏仁核（BAC1）、内嗅皮层（BAC5）、前额皮质（BAC4）、前脑中间神经元（BAC6）、腹侧被皮层（BAC8）和伏隔核（BAC3和BAC10）。到2004年9月，在转基因核心设施（Dr. James Pickel）的帮助下，我们完成了将HSP70最小启动子cre cDNA的DNA片段和每个BAC克隆的DNA片段共注射到小鼠受精卵中，产生双整合的转基因系。从那时起，我们花了大量的时间进行基因分型，并通过与Rosa26报告系LacZ杂交分析Cre重组模式。然而，在BAC克隆中产生的Cre/BAC双转基因系比预期的要少。在我们从Core Facility获得的106个Cre阳性转基因品系中，只有22个品系BAC阳性。双转基因小鼠产量低的原因可能是由于这种长度不同的DNA片段难以制备相同拷贝数。虽然我们仍在分析这22个系中的4个，但我们没有从它们中获得任何模拟Cre重组酶表达到每个BAC基因内源性表达的理想系。[我们还分析了超过50条cre阳性（BAC阴性）线，但没有一条像预期的那样有用]。尽管如此，我们已经建立了Cre系#4688，携带编码BAC1中降钙素受体样受体的DNA片段，其中Cre重组主要发生在齿状苔藓细胞和部分海马CA3c锥体细胞中。齿状苔藓细胞是一种谷氨酸能兴奋细胞，它接受来自齿状颗粒细胞的苔藓纤维并投射回颗粒细胞，在齿状回形成失突触的复发通路。在颞叶癫痫后发现了苔藓细胞的丢失和苔藓纤维的萌发。因此，Cre line #4688可用于齿状回功能和颞叶癫痫的研究。

The enormous complexity of the brain is derived from hundreds of neuronal cell types and extensive synaptic connections between them. Studies of the localized function of the brain subregions have recently been revolutionized by the development of genetic engineering that ideally switches gene expression on and off in a particular cell-type of a certain brain subregion in vivo. For example, Cre recombinase of the P1 bacteriophage has proven invaluable for conditional transgenic manipulation in post-mitotic neuronal cells of the adult brain. Since March 2003, we have initiated a project to create a variety of brain-subregion or cell-type restricted conditional transgenic mice. Our goal this year was to create various subregion-restricted Cre-recombinase transgenic lines, using a BAC (Bacterial Artificial Chromosome) clone carrying a promoter for directing the expression in particular brain areas. From the literature and by our in situ hybridization histochemistry, we identified some BAC clones that carry promoters for the gene expression predominantly in the hippocampal CA1 (namely BAC7), amygdala (BAC1), entorhinal cortex (BAC5), prefrontal cortex (BAC4), forebrain interneurons (BAC6), ventral tegmental area (BAC8) and nucleus accumbens (BAC3 and BAC10), respectively. By September 2004, with the help of the Transgenic Core Facility (Dr. James Pickel), we had finished co-injecting the DNA fragments of HSP70 minimal promoter-Cre cDNA and DNA fragment for each BAC clone into mouse fertilized eggs to generate doubly-integrated transgenic lines. Since then we have spent much time on genotyping and analyzing the Cre recombination pattern by crossing with Rosa26 reporter LacZ line. However, fewer Cre/BAC-double transgenic lines across BAC clones were generated than expected. Out of 106 Cre positive transgenic lines we obtained from the Core Facility, only 22 lines were also BAC positive. The reason for the low yield of double transgenic mice may be due to the difficulty in preparing the same copy number of DNA fragments of such different lengths. While we are still analyzing 4 of these 22 lines, we did not obtain any ideal lines that mimic the Cre recombinase expression to the endogenous expression of each BAC gene from them. [We also analyzed over 50 Cre-positive (BAC negative) lines, but none of them were as useful as expected]. Nevertheless, we have established a Cre line #4688, carrying the DNA fragment encoding calcitonin receptor-like receptor in BAC1, in which Cre recombination occurs predominantly in the dentate mossy cells and, in part, hippocampal CA3c pyramidal cells. The dentate mossy cell is a glutamatergic excitatory cell, which receives mossy fibers from dentate granule cells and projects back to granule cells, forming disynaptic recurrent pathway in the dentate gyrus. Mossy cell loss as well as mossy fiber sprouting has been recognized after temporal lobe epilepsy. Therefore, Cre line #4688 could be useful for the study of dentate gyrus function and temporal lobe epilepsy. In order to achieve the cell-type restricted Cre expression more efficiently, we have investigated several BAC manipulation methods since October 2004, and a new approach using RED/ET system has been employed to introduce Cre cDNA just prior to the initial methionine codon in the BAC clone, to obtain a single DNA fragment that includes the promoter region and Cre cDNA. Moreover, to achieve homologous recombination in any desired BAC clones, we introduced a SacB negative selection marker (a gift from Dr. Heintz lab at Rockefeller University) to exclude the shuttle vector backbone. This BAC manipulation method for homologous recombination now enables us to efficiently introduce Cre recombinase cDNA to any desired sites of the BAC clones. We have chosen 5 additional BAC clones, each of which contain the gene product whose expression is well confined to particular brain areas as mentioned above. Currently, BAC 14-Cre for prefrontal cortex, BAC16-Cre for VTA, and BAC 20-Cre for NAcc have been injected into eggs to generate Cre transgenic mice in the Transgenic Core Facility. Cre cDNA insertion by homologous recombination into other BAC clones is also underway. Since all the lines carry BAC-derived enhancer/promoter elements and Cre cDNA, it is probable for us to obtain cell-type or region-restricted Cre transgenic mouse lines dependent upon the target gene promoter in the BAC fragment. Once Cre-lines are established from these founder mice, we crossed them with a Rosa26 reporter line, in which the expression of Cre recombinase is functionally visualized by X-gal staining. Once these lines are established, we will further narrow down this project to target the NMDA receptor knockout to particular cell types and investigate the behavioral and physiological consequence of region-restricted knockout of NMDA receptors (NRs). The behavioral and physiological analyses of these future NR knockout mice may hopefully lead to our understanding of the most serious neuropsychiatric disorders, such as bipolar disorder and schizophrenia. We have also initiated two additional floxed mouse projects to explore the in vivo function of particular brain areas. One is to generate transgenic mice, in which cell ablation can be induced in the cells where Cre recombinase is highly expressed, using a variant of human heparin binding-epidermal growth factor (HB-EGF) precursor as a diphtheria toxin receptor (DTR). Since diphtheria toxin administered by intra-peritoneal injection does not bind to murine HB-EGF precursor, cell ablation by DT binding will depend on the expression of human HB-EGF following Cre-loxP excision in the floxed HB-EGF alleles of mice. In collaboration with Dr. Kenji Kohno in Nara, Japan, Kazu Nakazawa and Yoko Yabe have engaged in making the construct for floxed-human HB-EGF mice. Also, we have introduced a floxed mice carrying simian HB-EGF targeted to Rosa26, the house keeping gene locus, from Drs. Ari Waisman and Thorsten Buch in Germany. We hope to cross these floxed-DTR lines with future Cre lines. The second project to generate new floxed mice targets the Kir3.2 locus. In some brain areas, such as VTA, Kir3.2-type inwardly rectified potassium channels are predominantly expressed among many other Kir channels. Based on the report showing slight membrane depolarization of VTA in the Kir3.2 KO mice, we plan to generate floxed-Kir3.2 mice, and to cross with future VTA-Cre line to depolarize VTA, thereby altering dopamine secretion into NAcc. Juan Belforte and Noelia Vargas Pinto are working on making this knock-in construct of floxed-Kir3.2. This project, in conjunction with generation of VTA-Cre line, would provide an excellent tool for the study of dopamine function in the NAcc during behavior and in vivo physiology. Finally, it is possible to use genetic protein synthesis knock-down mice with future Cre lines, in order to understand the role of protein synthesis in the particular brain areas, as described in a separate Project (# MH002846-02) from my laboratory. These additional lines of floxed mice will enable us to investigate functional roles of particular brain areas in learning and memory at a different level.