Precise and in situ genetic humanization of 6 Mb of mouse immunoglobulin genes
Precise and in situ genetic humanization of 6 Mb of mouse immunoglobulin genes
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DOI：
10.1073/pnas.1323896111
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发表时间：
2014-04-08
影响因子：
11.1
通讯作者：
Murphy, Andrew J.
中科院分区：
文献类型：
--
作者：
Macdonald, Lynn E.;Karow, Margaret;Murphy, Andrew J.
基因人源化，包括用人类基因替换小鼠基因，可以为人类基因和疾病的研究创造强大的动物模型。遗传人源化的一个重要实例涉及针对其IG基因进行人源化的小鼠，从而允许在小鼠背景（HumAb小鼠）内的人抗体应答，并且还提供用于产生作为治疗剂的完全人抗体的有价值的平台。然而，现有的HumAb小鼠不具有完全功能性的免疫系统，这可能是因为它们被基因人源化的方式。因此，大多数遗传人源化涉及内源小鼠基因的破坏，同时在新的和随机的位置引入人转基因（所谓的KO+转基因人源化）。最近的努力试图在相同的遗传位置用人类对应物取代小鼠基因（原位人源化），但这些努力涉及费力的程序，并且在大小和精度方面受到限制。我们描述了一个通用的和有效的方法，非常大的，原位，和精确的基因人源化使用大型复合细菌人工染色体为基础的靶向载体引入小鼠ES细胞。我们应用这种方法对小鼠重链和小鼠巨噬细胞的3-Mb片段进行了基因人源化。轻链IG基因座，迄今为止所描述的最大的遗传人源化。本文详细描述了我们的遗传人源化方法，配套文件报告说，携带这些基因人源化位点的小鼠的体液免疫系统的功能与WT小鼠一样有效。
Genetic humanization, which involves replacing mouse genes with their human counterparts, can create powerful animal models for the study of human genes and diseases. One important example of genetic humanization involves mice humanized for their Ig genes, allowing for human antibody responses within a mouse background (HumAb mice) and also providing a valuable platform for the generation of fully human antibodies as therapeutics. However, existing HumAb mice do not have fully functional immune systems, perhaps because of the manner in which they were genetically humanized. Heretofore, most genetic humanizations have involved disruption of the endogenous mouse gene with simultaneous introduction of a human transgene at a new and random location (so-called KO-plus-transgenic humanization). More recent efforts have attempted to replace mouse genes with their human counterparts at the same genetic location (in situ humanization), but such efforts involved laborious procedures and were limited in size and precision. We describe a general and efficient method for very large, in situ, and precise genetic humanization using large compound bacterial artificial chromosome-based targeting vectors introduced into mouse ES cells. We applied this method to genetically humanize 3-Mb segments of both the mouse heavy and. light chain Ig loci, by far the largest genetic humanizations ever described. This paper provides a detailed description of our genetic humanization approach, and the companion paper reports that the humoral immune systems of mice bearing these genetically humanized loci function as efficiently as those of WT mice.