Precise and in situ genetic humanization of 6 Mb of mouse immunoglobulin genes

Abstract

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 κ 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.genome engineering | therapeutic antibody | immunoglobulin locus T he laboratory mouse is one of the premier model organisms used by biologists. As a mammal, the mouse is more genetically similar to humans and thus more relevant to human physiology and disease than many other model organisms. Its small size, short generation time, and the availability of a large variety of inbred strains have led to a robust body of classical genetic research on the mouse. The utility of mice as a genetic model is also greatly enhanced by powerful transgenic and knockout technologies, allowing researchers to study the effects of the directed overexpression or deletion of specific genes. However, despite all of its advantages, the mouse remains an imperfect model of human disease and an imperfect platform on which to test potential human therapeutics. One issue is that, although about 99% of human genes have a mouse homolog (1), potential therapeutic agents often do not cross-react, or cross-react only poorly, with the mouse ortholog of the intended human target. To overcome this problem, selected target genes can be humanized, that is, the mouse gene can be eliminated and replaced by the corresponding human orthologous gene sequence. Because of the difficulties of using conventional KO technologies to directly replace large mouse genes with their large human genomic counterparts, genetic humanization is currently mo...

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