Morton Magda F, Liu Pei-Qi, Reik Andreas, de la Rosa Ragan, Mendel Matthew, Li Xiao-Yong, Case Casey, Pabo Carl, Moreno Veronica, Pyati Jayashree, Shankley Nigel P
Johnson & Johnson Pharmaceutical Research & Development, LLC., 3210 Merryfield Row, San Diego, CA, 92121, USA.
Regul Pept. 2005 Jul 15;129(1-3):227-32. doi: 10.1016/j.regpep.2005.02.013.
Designed zinc finger proteins (ZFPs) regulate expression of target genes when coupled to activator or repressor domains. Transfection of ZFPs into cell lines can create expression systems where the targeted endogenous gene is transcribed and the protein of interest can be investigated in its own cellular context. Here we describe the pharmacological investigation of an expression system generated using CCK2 receptor-selective ZFPs transfected into human embryonic kidney cells (HEKZFP system). The receptors expressed in this system, in response to ZFP expression, were functional in calcium mobilization studies and the potency of the agonists investigated was consistent with their action at CCK2 receptors (CCK-8S pA50 = 9.05+/-0.11, pentagastrin pA50 = 9.11+/-0.13). In addition, binding studies were conducted using [125I]-BH-CCK-8S as radioligand. The saturation binding analysis of this radioligand was consistent with a single population of high affinity CCK receptors (pK(D) = 10.24). Competition studies were also conducted using a number of previously well-characterized CCK-receptor selective ligands; JB93182, YF476, PD-134,308, SR27897, dexloxiglumide, L-365,260 and L-364,718. Overall, the estimated affinity values for these ligands were consistent with their interaction at CCK2 receptors. Therefore, CCK2 receptors up-regulated using zinc finger protein technology can provide an alternative to standard transfection techniques for the pharmacological analysis of compounds.
设计的锌指蛋白（ZFPs）与激活域或抑制域偶联时可调节靶基因的表达。将锌指蛋白转染到细胞系中可创建表达系统，在此系统中，靶向的内源性基因被转录，并且可以在其自身的细胞环境中研究感兴趣的蛋白质。在此，我们描述了对使用转染到人胚肾细胞中的CCK2受体选择性锌指蛋白（HEKZFP系统）生成的表达系统进行的药理学研究。在该系统中表达的受体，响应锌指蛋白的表达，在钙动员研究中具有功能，并且所研究的激动剂的效力与其在CCK2受体上的作用一致（CCK-8S的pA50 =9.05±0.11；五肽胃泌素的pA50 =9.11±0.13）。此外，使用[125I]-BH-CCK-8S作为放射性配体进行了结合研究。该放射性配体的饱和结合分析与单一群体的高亲和力CCK受体一致（pK(D)= 10.24）。还使用了许多先前已充分表征的CCK受体选择性配体进行了竞争研究；JB93182、YF476、PD-134308、SR27897、右旋洛谷酰胺、L-365260和L-364718。总体而言，这些配体的估计亲和力值与其在CCK2受体上的相互作用一致。因此，使用锌指蛋白技术上调的CCK2受体可为化合物的药理学分析提供一种替代标准转染技术的方法。
