A well-differentiated B-cell line is permissive for somatic mutation of a transfected immunoglobulin heavy-chain gene.

pSV2neo plasmids containing an IgM heavy-chain gene with nonsense mutations in either the variable (V) or the constant (C) region were transfected into four differentiated mouse plasma cell lines: S107 and the NSO fusion partner (myeloma cell lines) and 2C3 and 36.65 (hybridoma cell lines). The frequencies of reversion of the nonsense mutations in multiple independent transfectants were determined with the spot ELISA and rates of reversion were calculated by fluctuation analysis. Mutations in both V and C regions were confirmed by sequence analyses. In the S107 cell line, spontaneous point mutations occurred in the V region at a rate of approximately 5 x 10(-5)/bp per cell generation, > 400-fold higher than the rate of V-region mutation in the NSO cell line and considerably higher than the rates in 2C3 and 36.65 hybridoma cell lines. These studies suggest that S107 is a relatively permissive cell line in which V-region mutations can occur constitutively, even though it represents a late stage of B-cell differentiation. Further, the results show that the construct used contains sufficient information in its flanking and coding sequences to allow a relatively high rate of V-region mutation, at least in the S107 cell line. IMAGES:

将携带在可变区（V）或恒定区（C）带有无义突变的IgM重链基因的pSV2neo质粒转染至四株分化成熟的小鼠浆细胞系：其中S107与NSO融合株属于骨髓瘤细胞系（myeloma cell lines），2C3和36.65属于杂交瘤细胞系（hybridoma cell lines）。通过斑点酶联免疫吸附试验（spot ELISA）对多株独立转染子的无义突变回复频率进行检测，并通过波动分析（fluctuation analysis）计算回复突变率。通过序列分析（sequence analyses）验证了V区与C区的突变。在S107细胞系中，可变区的自发点突变率约为每细胞世代5×10^(-5)/碱基对（base pair，bp），较NSO细胞系的可变区突变率高出400倍以上，且显著高于2C3与36.65杂交瘤细胞系的突变水平。本研究表明，S107是一种相对允许性的细胞系，即便其处于B细胞分化的晚期阶段，可变区突变仍可组成型发生。此外，实验结果显示，本次研究所用的构建体在其侧翼序列与编码序列中携带了足够的遗传信息，可介导较高水平的可变区突变——至少在S107细胞系中如此。IMAGES: