Mocikat R, Kardinal C, Selmayr M
GSF Institut für Immunologie, München, Germany.
Mol Gen Genet. 1997 Nov;256(5):499-508. doi: 10.1007/s004380050594.
The site-specific integration of exogenous gene fragments by homologous recombination provides a convenient method for altering the immunoglobulin loci of B cells and specifically designing antibody molecules. To introduce a human isotype into the heavy chain locus of mouse hybridoma cells we compared the recombination frequencies of vectors that could be linearized either as integration or as replacement constructs in different cell lines. Integration as well as replacement recombination was observed, irrespective of the location of the site at which the vector was cleaved. Integration events involving the human IgG1 vectors were lost at high frequency due to secondary vector excision, so that all stable recombinations were found to be replacement events. Replacement recombination of an integration vector involves an illegitimate crossover at least at the 3' side and sometimes gives rise to deletion of the CH1 domain. However, a homologous event at the 3' side is more efficient than an illegitimate one, so that a homology that is distributed on both sides of the heterologous region promotes targeting at higher frequency than a contiguous sequence of the same total length. The position of the linearization site in the vector markedly influenced the targeting efficiency, but surprisingly, whether a double-strand break in the homology or in the heterology region more efficiently promoted integration was dependent on the cell line. In all cells, however, cleavage of the vector outside the homology region favoured stable replacements with a bias against CH1-truncated clones. We further show that the frequency of replacements induced by integration vectors is not correlated to the homology length and cannot be increased by irradiation of the cells. Our findings indicate that for targeting the IgH locus other mechanisms might be involved than at other loci.
通过同源重组实现外源基因片段的位点特异性整合，为改变B细胞的免疫球蛋白基因座和特异性设计抗体分子提供了一种便捷方法。为了将人免疫球蛋白同种型引入小鼠杂交瘤细胞的重链基因座，我们比较了在不同细胞系中可作为整合构建体或置换构建体线性化的载体的重组频率。无论载体切割位点的位置如何，均观察到整合以及置换重组。由于二次载体切除，涉及人IgG1载体的整合事件高频丢失，因此所有稳定重组均为置换事件。整合载体的置换重组至少在3'端涉及非法交叉，有时会导致CH1结构域缺失。然而，3'端的同源事件比非法事件更有效，因此分布在异源区域两侧的同源性比相同总长度的连续序列更能促进更高频率的靶向。载体中线性化位点的位置显著影响靶向效率，但令人惊讶的是，同源区域或异源区域中的双链断裂更有效地促进整合取决于细胞系。然而，在所有细胞中，在同源区域外切割载体有利于稳定置换，且偏向于不含CH1截短克隆。我们进一步表明，整合载体诱导的置换频率与同源长度无关，并且不能通过照射细胞来提高。我们的研究结果表明，对于靶向IgH基因座，可能涉及与其他基因座不同的机制。
