Molecular evolution system of proteins using a B cell line with spontaneous mutation machinery

鸡B淋巴瘤系DT40在培养过程中自发地高突变免疫球蛋白(Ig)基因。因此，培养的DT40细胞构成了一个有用的Ig文库，用于体外筛选抗体(Abs)。为了通过停止超突变来获得理想的Ig突变体，或者为了进一步提高抗体水平而恢复突变，激活诱导的胞苷脱氨酶(AID)是负责Ig突变机制的关键酶，必须被打开或关闭。为此，我们产生了一个DT40品系，它的一个AID等位基因被破坏，另一个等位基因被loxP-.anked AID结构所取代。在这个命名为DT40-SW的工程细胞系中，AID的表达可以被他莫昔芬调节的Cre重组酶可逆地转换。此外，还引入了通过绿色荧光蛋白表达和嘌呤霉素抗性来区分“AID-ON”和“AID-OFF”细胞的装置。从单个DT40-SW细胞开始，只有当Aid表达开启时，Ig基因库才能在培养过程中有效地多样化。We…以绿色荧光蛋白(GFP)基因为供体，蓝色荧光蛋白(BFP)基因为受体的基因转化底物的DT4O品系较多。当底物结构整合到免疫球蛋白L链上时，表达bfp的细胞将其荧光从蓝色转换为绿色。因此，DT4O细胞中的基因转换机制将有助于设计非Ig蛋白。为了研究生发中心(GC)反应的机制，我们建立了一种命名为PFL的细胞系，该细胞系保留了小鼠淋巴结节段三维培养的主要FDC表型。β受体激动型抗淋巴毒素受体(LT-βR)单抗和α刺激后，PFL细胞增殖缓慢。从PFL的长期培养中分离到一个生长较快的克隆FL-Y。对这两种细胞系表面标志物的分析揭示了FDDC特有的基因表达。因此，PFL和FL-Y细胞可能有助于深入了解FDC在GC减少中的功能作用

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.x desirable Ig mutants by stopping hypermutation or to resume mutation for further improvement of Ab a.nity, activation-induced cytidine deaminase (AID), a key enzyme responsible for the Ig mutation machinery, must be switched on or o.. To this end, we generated a DT40 line whose one AID allele was disrupted, and the other allele was replaced by the loxP-.anked 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.We … More generated DT4O lines that bears a gene conversion substrate comprising the green fluorescent protein (GFP) gene as a donor and the blue fluorescent protein (BFP) gene as an acceptor. BFP-expressing cells converted their fluorescence from blue to green when the substrate construct was integrated in the Ig L chain locus. Thus, the gene conversion machinery in DT4O cells will be useful to engineer non-Ig proteins.To investigate mechanisms underlying germinal center (GC) reaction, we established a cell line designated as pFL that retained major FDC phenotypes from a 3-dimensional culture of mouse lymph node segments. pFL cells proliferated slowly in response to an agonistic anti-lymphotoxin β receptor (LTβR) mAb and TNFα. A more rapidly growing clone, named FL-Y was isolated from a long-term culture of pFL. Analysis of surface markers in these two cell lines revealed the expression of genes that are characteristic of FDCs. Thus, pFL and FL-Y cells may be useful for providing insight into the functional role for FDC in GC Less