Batrak Vera, Blagodatski Artem, Buerstedde Jean-Marie
Methods Mol Biol. 2011;745:311-26. doi: 10.1007/978-1-61779-129-1_18.
The immunoglobulin (Ig) genes of B cells are diversified at high rate by point mutations whereas the non-Ig genes of B cells accumulate no or significantly fewer mutations. Ig hypermutations are critical for the affinity maturation of antibodies for most of jawed vertebrates and also contribute to the primary Ig diversity repertoire formation in some species. How the hypermutation activity is specifically targeted to the Ig loci is a long-standing debate. Here we describe a new experimental approach to investigate the locus specificity of Ig hypermutation using the chicken B-cell line DT40. One feature is the use of a green fluorescent protein (GFP) gene as a mutation reporter. Some nucleotide changes produced by somatic hypermutation can cripple the GFP gene which leads to a decrease or loss of the green fluorescence. Therefore such changes can be easily quantified by fluorescence-activated cell sorting (FACS). Another advantage of this approach is the targeted integration of the mutation reporter into a defined chromosomal position. This system allowed us to identify a 10 kb sequence within the Ig light chain (IgL) locus, which is both necessary and sufficient to activate hypermutation in the neighboring reporter gene. We have called this sequence Diversification Activator (DIVAC) and postulated that similar cis-acting sequences exist in the heavy and light chain Ig loci of all jawed vertebrate species. Our experimental system promises further insight into the molecular mechanism of Ig hypermutation. For example, it may be possible to identify smaller functional motifs within DIVAC and address the role of putative transacting binding factors by gene knock-outs.
B细胞的免疫球蛋白（Ig）基因通过点突变以高频率多样化，而B细胞的非Ig基因积累的突变很少或几乎没有。Ig超突变对于大多数有颌脊椎动物抗体的亲和力成熟至关重要，并且在某些物种中也有助于初级Ig多样性库的形成。超突变活性如何特异性地靶向Ig基因座一直是一个长期争论的问题。在这里，我们描述了一种新的实验方法，使用鸡B细胞系DT40来研究Ig超突变的基因座特异性。一个特点是使用绿色荧光蛋白（GFP）基因作为突变报告基因。体细胞超突变产生的一些核苷酸变化会使GFP基因失活，导致绿色荧光减弱或消失。因此，这种变化可以通过荧光激活细胞分选（FACS）轻松定量。这种方法的另一个优点是将突变报告基因靶向整合到确定的染色体位置。该系统使我们能够在Ig轻链（IgL）基因座内鉴定出一个10 kb的序列，该序列对于激活邻近报告基因中的超突变既必要又充分。我们将这个序列称为多样化激活因子（DIVAC），并推测在所有有颌脊椎动物物种的重链和轻链Ig基因座中都存在类似的顺式作用序列。我们的实验系统有望进一步深入了解Ig超突变的分子机制。例如，有可能在DIVAC内鉴定出更小的功能基序，并通过基因敲除来研究推定的反式作用结合因子的作用。
