Reversible switching of immunoglobulin hypermutation machinery in a chicken B cell line
Reversible switching of immunoglobulin hypermutation machinery in a chicken B cell line
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DOI：
10.1016/j.bbrc.2004.11.143
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发表时间：
2005-02-04
影响因子：
3.1
通讯作者：
Ohmori, H
中科院分区：
文献类型：
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作者：
Kanayama, N;Todo, K;Ohmori, H
鸡B淋巴瘤系DT40在培养过程中自发地产生免疫球蛋白（Ig）基因高突变。因此，培养的DT - 40细胞构成了一个有用的体外筛选抗体（Abs）的Ig文库。为了通过停止高突变或恢复突变来修复所需的Ig突变体以进一步改善Ab亲和力，必须打开或关闭激活诱导胞苷脱氨酶（AID），这是负责Ig突变机制的关键酶。为此，我们产生了一个DT40系，其中一个AID等位基因被破坏，另一个等位基因被loxp侧翼的AID结构所取代。在这个被命名为DT40-SW的工程细胞系中，AID的表达可以被他莫昔芬调节的Cre重组酶可逆地改变。还引入了分别通过GFP表达和嘌呤霉素耐药性来区分“aids - on”和“aids - off”细胞的装置。从单个DT40-SW细胞开始，只有在AID表达开启的情况下，Ig基因库才能在培养过程中有效地多样化。(C) 2004爱思唯尔公司版权所有。
A chicken B lymphoma line, DT40, hypermutates immunoglobulin (Ig) genes spontaneously during culture. Thus, cultured DT 40 cells constitute a useful Ig library for screening antibodies(Abs) in vitro. To fix desirable Ig mutants by stopping hypermutation or to resume mutation for further improvement of Ab affinity, activation-induced cytidine deaminase (AID), a key enzyme responsible for the Ig mutation machinery, must be switched on or off. To this end, we generated a DT40 line whose one AID allele was disrupted, and the other allele was replaced by the loxP-flanked AID construct. In this engineered cell line designated as DT40-SW, AID expression could be switched reversibly by tamoxifen-regulated Cre recombinase. Devices were also introduced to discriminate between the "AID-ON" and the "AID-OFF" cells by GFP expression and puromycin resistance, respectively. Starting from a single DT40-SW cell, Ig gene repertoire was efficiently diversified during culture only when AID expression was on. (C) 2004 Elsevier Inc. All rights reserved.