Kardinal C, Hooijberg E, Lang P, Zeidler R, Mocikat R
GSF-Institut für Immunologie, München, Germany.
Eur J Immunol. 1995 Mar;25(3):792-7. doi: 10.1002/eji.1830250325.
Gene targeting in hybridoma cells provides a tool for generating chimeric antibodies with great ease and at high yield. We present an evaluation of integration vectors for the chimerization of the immunoglobulin heavy chain locus which are universally applicable to hybridomas of different isotypes and mouse strains. There are three problems arising with vector integration: (i) the frequent persistence of the parental isotype; (ii) an isotype-dependent aberrant replacement-like recombination giving rise to antibodies devoid of the CH1 domain; and (iii) secondary recombinations leading to excision of the integrated sequence. To overcome these problems, we have systematically evaluated the consequences of extending the vector flank. Although the homology length clearly determines the recombination frequency, this effect is counteracted by the secondary recombination, which also correlates to the homology length. In contrast, the truncating recombination events are not dependent on the homology length and never lead to re-excision of the construct. To take advantage of the increased genetic stability obtained with short flanks, we constructed an enrichment vector which yields high recombination efficiencies despite using a short flanking sequence. In addition, irradiation of the cells enhanced homologous recombination. The problem of the co-production of two isotypes was overcome by a two-step targeting reaction.
杂交瘤细胞中的基因靶向技术为轻松且高产地产生活嵌合抗体提供了一种工具。我们对用于免疫球蛋白重链基因座嵌合化的整合载体进行了评估，这些载体普遍适用于不同同种型和小鼠品系的杂交瘤。载体整合会出现三个问题：（i）亲本同种型频繁持续存在；（ii）同种型依赖性异常替代样重组导致缺乏CH1结构域的抗体产生；（iii）二次重组导致整合序列切除。为克服这些问题，我们系统评估了延长载体侧翼的后果。虽然同源长度明显决定重组频率，但这种效应会被二次重组抵消，二次重组也与同源长度相关。相比之下，截断性重组事件不依赖于同源长度，且从不导致构建体重新切除。为利用短侧翼获得的更高遗传稳定性，我们构建了一种富集载体，尽管使用短侧翼序列，但仍能产生高重组效率。此外，细胞照射增强了同源重组。通过两步靶向反应克服了两种同种型共同产生的问题。
