Optogenetics and Transgenic Technology Core

我们的工作人员精通包装、纯化和鉴定(体外和体内)腺相关病毒(AAV)、慢病毒载体(LV)和犬腺病毒载体(CAV2)的方法。我们经常为NIDA IRP以及其他校内和校外合作生成载体。这些载体主要是为了研究成瘾和神经退化的分子机制而设计的。例如，我们已经生产了40多种不同的载体，用于递送视蛋白，用于神经元电路的光遗传操作。我们还开发了几个新的载体来表达突触靶向荧光蛋白、钙敏感荧光蛋白、红外报告蛋白和光敏蛋白，用于光损伤神经元。 我们已经成功地创建了16个转基因大鼠系，但尚未发表，其中包括一个条件-mCherry荧光报告鼠，它只在Cre重组酶存在的情况下表达mCherry荧光蛋白。这只大鼠已经与我们的DAT-Cre大鼠杂交，以验证Cre在多巴胺能神经元中的表达。我们还有一个表达Tet阻遏蛋白的转基因大鼠株系。Tet抑制蛋白可以结合特定的DNA元件(Tet操纵子)来阻止基因表达。通过给动物服用四环素或类似物，我们可以使抑制物脱离DNA，允许基因表达。这将与含有TET操作符的病毒或其他大鼠结合使用，以允许我们激活基因表达。我们还有更多的CRE驱动大鼠处于早期特征阶段，以及其他报告大鼠。 我们所有的特色化和发布的载体都可供校内和校外的合作者使用。

Our staff are specialized 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 40 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 16 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.