Holst Jeff, Vignali Kate M, Burton Amanda R, Vignali Dario A A
Department of Immunology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, Tennessee 38105, USA.
Nat Methods. 2006 Mar;3(3):191-7. doi: 10.1038/nmeth858.
Although T-cell receptor (TCR) transgenic as well as knockout and knockin mice have had a large impact on our understanding of T-cell development, signal transduction and function, the need to cross these mice delays experiments considerably. Here we provide a methodology for the rapid expression of TCRs in mice using 2A peptide-linked multicistronic retroviral vectors to transduce stem cells of any background before adoptive transfer into RAG-1(-/-) mice. For simplicity, we refer to these as retrogenic mice. We demonstrate that these retrogenic mice are comparable to transgenic mice expressing three commonly used TCRs (OT-I, OT-II [corrected] and AND). We also show that retrogenic mice expressing male antigen-specific TCRs (HY, MataHari and Marilyn) facilitated the analysis of positive and negative selection in female and male mice, respectively. We examined various tolerance mechanisms in epitope-coupled TCR retrogenic mice. This powerful resource could expedite the identification of proteins involved in T-cell development and function.
尽管T细胞受体（TCR）转基因小鼠以及基因敲除和基因敲入小鼠对我们理解T细胞发育、信号转导和功能产生了重大影响，但将这些小鼠进行杂交的需求大大延迟了实验进程。在此，我们提供一种方法，利用2A肽连接的多顺反子逆转录病毒载体在小鼠中快速表达TCR，以便在将干细胞过继转移到RAG-1（-/-）小鼠之前转导任何背景的干细胞。为简便起见，我们将这些小鼠称为逆转录基因小鼠。我们证明这些逆转录基因小鼠与表达三种常用TCR（OT-I、OT-II[校正后]和AND）的转基因小鼠相当。我们还表明，表达雄性抗原特异性TCR（HY、玛塔·哈里和玛丽莲）的逆转录基因小鼠分别有助于分析雌性和雄性小鼠中的阳性和阴性选择。我们研究了表位偶联TCR逆转录基因小鼠中的各种耐受机制。这种强大的资源可以加快对参与T细胞发育和功能的蛋白质的鉴定。
