Morran Michael P, Al-Dieri Ali G, Nestor-Kalinoski Andrea L, Jordan Richard K, Gupta Nirdesh K, McInerney Marcia F
Department of Medicinal and Biological Chemistry, College of Pharmacy and Pharmaceutical Sciences, University of Toledo, Toledo, OH 43606, USA.
Department of Surgery, College of Medicine and Life Sciences, University of Toledo, Toledo, OH 43606, USA.
J Biol Methods. 2018;5(1). doi: 10.14440/jbm.2018.209. Epub 2018 Jan 23.
The insulin receptor (IR) is a transmembrane receptor which recognizes and binds the hormone insulin. We describe two models that were devised to explore the role of IR over-expression on T-lymphocytes and their chemotactic motility in the progression of type 1 diabetes. FVB/NJ-CD3-3×FLAG-mIR/MFM mice were generated to selectively over-express 3×FLAG tagged murine IR in T-lymphocytes an engineered CD3 enhancer and promoter construct. Insertion of the 3×FLAG-mIR transgene into FVB/NJ mice, a known non-autoimmune prone strain, lead to a minor population of detectable 3×FLAG-mIR tagged T-lymphocytes in peripheral blood and the presence of a few lymphocytes in the pancreas of the Tg+/- compared to age matched Tg-/- control mice. In order to induce stronger murine IR over-expression then what was observed with the CD3 enhancer promoter construct, a second system utilizing the strong CAG viral promoter was generated. This system induces cell specific IR over-expression upon Cre-Lox recombination to afford functional 3×FLAG tagged murine IR with an internal eGFP reporter. The pPNTlox2-3×FLAG-mIR plasmid was constructed and validated in HEK-Cre-RFP cells to ensure selective Cre recombinase based 3×FLAG-mIR expression, receptor ligand affinity towards insulin, and functional initiation of signal transduction upon insulin stimulation.
胰岛素受体（IR）是一种跨膜受体，可识别并结合激素胰岛素。我们描述了两种模型，旨在探讨IR过表达在1型糖尿病进展过程中对T淋巴细胞及其趋化运动的作用。通过工程化的CD3增强子和启动子构建体，生成了FVB/NJ-CD3-3×FLAG-mIR/MFM小鼠，以在T淋巴细胞中选择性过表达3×FLAG标记的小鼠IR。将3×FLAG-mIR转基因插入FVB/NJ小鼠（一种已知的非自身免疫易感品系）后，与年龄匹配的Tg-/-对照小鼠相比，在外周血中可检测到少量3×FLAG-mIR标记的T淋巴细胞，且在Tg+/-小鼠的胰腺中存在少量淋巴细胞。为了诱导比CD3增强子启动子构建体所观察到的更强的小鼠IR过表达，构建了第二个利用强CAG病毒启动子的系统。该系统在Cre-Lox重组后诱导细胞特异性IR过表达，以提供具有内部eGFP报告基因的功能性3×FLAG标记的小鼠IR。构建了pPNTlox2-3×FLAG-mIR质粒并在HEK-Cre-RFP细胞中进行了验证，以确保基于选择性Cre重组酶的3×FLAG-mIR表达、受体对胰岛素的配体亲和力以及胰岛素刺激后信号转导的功能性启动。
