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

鸡B淋巴瘤细胞系DT 40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DT 40细胞构成了用于体外筛选抗体（Ab）的有用的IG文库。为了通过终止超突变获得所需的IG突变体或为了恢复突变以进一步改善抗体活性，必须开启或关闭激活诱导的胞苷脱氨酶（AID），其是负责IG突变机制的关键酶。为此，我们产生了DT 40系，其一个AID等位基因被破坏，另一个等位基因被loxP-连接的AID构建体取代。在这个被命名为DT 40-SW的工程细胞系中，AID表达可以通过他莫昔芬调节的Cre重组酶可逆地转换。还引入装置以分别通过GFP表达和嘌呤霉素抗性来区分“AID-ON”和“AID-OFF”细胞。从单个DT 40-SW细胞开始，只有当AID表达开启时，IG基因库才在培养过程中有效地多样化。 ...更多信息 产生携带基因转化底物的DT 40系，所述基因转化底物包含作为供体的绿色荧光蛋白（GFP）基因和作为受体的蓝色荧光蛋白（BFP）基因。当底物构建体整合到IG L链基因座中时，表达BFP的细胞将其荧光从蓝色转化为绿色。因此，在DT 4 O细胞中的基因转换机制将是有用的工程non-Ig proteins.To调查机制的基础生发中心（GC）reactions. To，我们建立了一个细胞系命名为pFL，保留主要FDC表型从小鼠淋巴结节段的三维培养。pFL细胞对激动性抗光敏素β受体（LTβR）mAb和TNFα的反应缓慢。从pFL的长期培养物中分离出一个生长更快的克隆，命名为FL-Y。在这两个细胞系的表面标志物的分析揭示了基因的表达，是特征性的FDC。因此，pFL和FL-Y细胞可能有助于深入了解FDC在GC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