Genetic manipulation of an exogenous non-immunoglobulin protein by gene conversion machinery in a chicken B cell line
Genetic manipulation of an exogenous non-immunoglobulin protein by gene conversion machinery in a chicken B cell line
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DOI：
10.1093/nar/gnj013
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发表时间：
2006-01-01
影响因子：
14.9
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
在培养过程中，鸡B细胞系DT 40通过基因转换自发地突变免疫球蛋白（IG）基因，该基因转换涉及激活诱导的胞苷脱氨酶（AID）依赖性可变（V）区基因与上游假V基因的同源重组。为了探索这种突变机制是否可以靶向外源非Ig基因，我们产生了携带基因转化底物的DT 40系，所述基因转化底物包括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。培养2-3周后，当底物构建体整合到IG轻链基因座而不是卵清蛋白基因座中时，几个百分比的最初表达BFP的细胞将其荧光从蓝色转化为绿色。这是BFP基因的AIDS依赖性和GFP基因模板化基因转化的结果，从而导致将各种大小的GFP衍生的基因片段引入BFP基因。因此，G/B构建体可用于可视化基因转化事件。在关闭DT 40细胞中的AID表达后，稳定分离突变克隆并保持其突变被固定。因此，DT 40细胞中的基因转换机制将是一种有用的手段，通过一种类型的DNA改组工程非Ig蛋白。
During culture, a chicken B cell line DT40 spontaneously mutates immunoglobulin (Ig) genes by gene conversion, which involves activation-induced cytidine deaminase (AID)-dependent homologous recombination of the variable (V) region gene with upstream pseudo-V genes. To explore whether this mutation mechanism can target exogenous non-Ig genes, we generated DT40 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. A few percent of the initially BFP-expressing cells converted their fluorescence from blue to green after culture for 2-3 weeks when the substrate construct was integrated in the Ig light chain locus, but not in the ovalbumin locus. This was the result of AID-dependent and the GFP gene-templated gene conversion of the BFP gene, thereby leading to the introduction of various sizes of GFP-derived gene segment into the BFP gene. Thus, G/B construct may be used to visualize gene conversion events. After switching off AID expression in DT40 cells, the mutant clones were isolated stably and maintained with their mutations being fixed. Thus, the gene conversion machinery in DT40 cells will be a useful means to engineer non-Ig proteins by a type of DNA shuffling.