AAV-based toolkit for targeting specific cell types

描述(由申请人提供)：我们的目标是开发一种高效且廉价的工具包，用于消融或可逆地灭活大脑或其他器官中特定类型的细胞 哺乳动物。我们提出了用腺相关病毒载体(AAV)将硝基还原酶(NTR)或果蝇合成抑素受体(ALSTR)基因导入细胞的新技术。两者都是与增强型绿色荧光蛋白(EGFP)相连的融合蛋白，因此很容易识别转基因细胞。然后，转基因细胞要么被IP切除。注射CB1954或局部注射别抑素使其失活。与光遗传学或使用抗体或配体结合的细胞毒素(如皂苷)消融细胞等相关技术相比，这些新方法提供了几个优点。这些好处包括细胞靶向的高选择性，试剂盒组件的相对较低的成本，以及不需要昂贵的设备。另一个优点是，AAVs可以有效地转化哺乳动物细胞，并可以在BSL1下使用，BSL1是大多数生物实验室批准的生物安全水平。有了这些优势，我们期待我们的工具箱将被用于许多实验室，以有效地测试不同类型细胞的生理功能。为了实现高选择性的细胞靶向，这些技术使用在细胞特异性启动子控制下表达Cre重组酶的转基因小鼠。Cre-Lox重组涉及靶向位于loxP位点两侧的特定DNA序列。这种重组可以拼接或颠倒loxP位点之间的DNA片段。在我们提出的研究中，Cre重组酶要么拼接出一个停止盒，要么将插入序列反转到正义方向，从而只允许外源基因在靶细胞中表达。该项目的具体目的是：1.构建能够在含有Cre重组酶的细胞中选择性表达硝基还原酶和别抑素受体的AAV载体。2.在表达Cre的小鼠的多个品系中测试所构建的AAV载体，并确定最有效的方案来消融或灭活含Cre的细胞。我们相信，由于该方法独特而强大的特点，我们的工具包将对研究界有很大的帮助。

DESCRIPTION (provided by applicant): Our goal is to develop an efficient and inexpensive toolkit for ablating or reversibly inactivating specific cell types in the brain or other organs of mammals. We propose new techniques in which the genes for nitroreductase (NTR) or Drosophila allatostatin receptor (AlstR) are delivered into cells using an adeno-associated viral vector (AAV). Both are fusion proteins linked to enhanced green fluorescent protein (EGFP), making it easy to identify transfected cells. The transfected cells are then either ablated by an i.p. injection of CB1954 or inactivated by a local injection of allatostatin. These new methods provide several advantages over related techniques such as optogenetics or ablation of cells using cytotoxins (e.g. saporin) conjugated to antibodies or ligands. These benefits include a high selectivity of cell targeting, a relatively low cost for the kit's components, and no requirement for expensive equipment. Another advantage is that AAVs efficiently transfect mammalian cells and can be used under BSL1, the biosafety level approved in most biological laboratories. With these advantages, we expect that our toolkit will be used in many labs to efficiently test the physiological functions of different cell types. To achieve high selectivity f cell targeting, these techniques use transgenic mice expressing Cre recombinase under control of cell-specific promoters. Cre-Lox recombination involves the targeting of specific sequences of DNA that are flanked by loxP sites. This recombination can splice out or invert the DNA fragment between the loxP sites. In our proposed studies, Cre recombinase will either splice out a STOP cassette or invert the insert sequence into the sense direction, allowing the expression of foreign genes only in targeted cells. The Specific Aims of the proposed project are to: 1. Construct AAV vectors that enable robust expression of nitroreductase and allatostatin receptor selectively in cells containing Cre recombinase. 2. Test the constructed AAV vectors in several lines of Cre-expressing mice and identify the most efficient protocol for ablation or inactivation f the Cre-containing cells. We believe that our toolkit will be of great help to the research community due to the unique and powerful features of the method.