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有效且廉价的工具包，用于消融或可逆灭活脑或其他器官中的特定细胞类型， 哺乳动物我们提出了新的技术，其中硝基还原酶（NTR）或果蝇allatostatin受体（AlstR）的基因被传递到细胞使用腺相关病毒载体（AAV）。两者都是与增强型绿色荧光蛋白（EGFP）连接的融合蛋白，使得识别转染细胞变得容易。然后通过腹膜内注射CB 1954消除转染的细胞或通过局部注射allatostatin灭活转染的细胞。 这些新方法提供了优于相关技术的几个优点，例如光遗传学或使用与抗体或配体缀合的细胞毒素（例如皂草素）的细胞消融。这些益处包括细胞靶向的高选择性、试剂盒组分的相对低的成本以及不需要昂贵的设备。另一个优点是AAV有效地感染哺乳动物细胞，并且可以在大多数生物实验室批准的生物安全水平BSL 1下使用。有了这些优势，我们希望我们的工具包将在许多实验室中使用，以有效地测试不同细胞类型的生理功能。 为了实现细胞靶向的高选择性，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重组涉及靶向侧翼为loxP位点的DNA的特定序列。这种重组可以剪接或反转loxP位点之间的DNA片段。在我们提出的研究中，Cre重组酶将剪接出STOP盒或将插入序列反转为有义方向，从而仅允许外源基因在靶细胞中表达。 拟议项目的具体目标是：1。构建能够在含有Cre重组酶的细胞中选择性地稳健表达硝基还原酶和allatostatin受体的AAV载体。2.在几种表达Cre的小鼠品系中测试构建的AAV载体，并鉴定用于消融或灭活含Cre细胞的最有效方案。 我们相信，由于该方法的独特和强大的功能，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.