Optogenetics and Transgenic Technology Core

我们现在拥有专门从事腺相关病毒（AAV）、慢病毒载体（LV）和犬腺病毒载体（CAV 2）包装、纯化和表征（体外和体内）方法的工作人员。 我们经常为NIDA IRP以及其他校内和校外合作生成矢量。 这些载体主要用于研究成瘾和神经退行性变的分子机制。 例如，我们已经生产了超过20种不同的载体，用于递送视蛋白，用于神经元回路的光遗传学操纵。 我们还开发了几种新的载体来表达突触靶向荧光蛋白、钙敏感荧光蛋白、红外报告蛋白和用于光损伤神经元的光敏蛋白。 我们已经成功地创建了12个转基因大鼠品系，但尚未发表，其中包括一个条件性mCherry荧光报告大鼠，该大鼠仅在Cre重组酶存在的情况下表达mCherry荧光蛋白。 该大鼠已与我们的DAT-Cre大鼠杂交以验证Cre在多巴胺能神经元中的表达。 我们也有一个转基因大鼠系表达泰特阻遏蛋白。 泰特阻遏蛋白可与特异性DNA元件（泰特操纵子）结合以阻止基因表达。通过给动物四环素或类似物，我们可以使阻遏物从DNA上脱落，使基因表达。这将与病毒或其他含有泰特操纵基因的大鼠联合使用，使我们能够激活基因表达。 我们在表征的早期阶段有额外的Cre-driver大鼠以及其他报告大鼠。 我们所有的特征和出版的载体可供校内和校外合作者。

We now have staff who are specilized in the methods to package, purify and characterize (in vitro and in vivo) both adeno-associated viral (AAV), lentiviral vectors (LV)and Canine adenoviral vectors (CAV2). We routinely generate vectors both for the NIDA IRP as well as other intramural and extramural collaborations. The vectors are designed primarily for studies focused on molecular mechanisms of addiction and neurodegeneration. For example, we have produced over 20 different vectors for delivery of the opsins for optogenetic manipulation of neuronal circuits. We have also been developing several novel vectors to express synaptically targeted fluorescent proteins, calcium-sensitive fluorescent proteins, infrared reporter protein and photosensitizing proteins for lesioning neurons with light. We have successfully created but not yet published 12 transgenic rat lines including a conditional-mCherry fluorescent reporter rat that will only express the mCherry fluorescent protein where Cre recombinase is present. This rat has been crossed with our DAT-Cre rat to verify expression of Cre in dopaminergic neurons. We also have a transgenic rat line expressing Tet repressor protein. The Tet repressor protein can bind to specific DNA elements (Tet operator) to prevent gene expression. By giving animals tetracycline or an analog we can cause the repressor to come off the DNA and allow gene expression. This will be used in combination with viruses or other rats that containing the Tet operator to allow us to activate gene expression. We have additional Cre-driver rats in early stages of characterization as well as other reporter rats. All of our characterized and published vectors are available for intramural and extramural collaborators.