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.

Keywords: genome engineering; immunoglobulin locus; therapeutic antibody.

Fig. 1.

Humanization of the mouse Ig heavy chain variable locus. Representations are not drawn to scale. The general scheme for direct genomic swap is depicted in A. The first three steps (B; steps A, B, and C) result in deletion of all mouse V_H, D_H, and J_H gene segments and insertion of all human D_H and J_H segments and three human V_H gene segments. The representative 3hV_H BACvec shown for step A consists of a 67-kb mouse homology arm, a neo selection cassette, a 145-kb human insert, and an 8-kb mouse homology arm. The next six steps (C) result in the insertion of the remainder of the human V gene segments and the removal of the final neo selection cassette. The representative 18hV_H BACvec for step D consists of a 20-kb mouse homology arm, a neo selection cassette, 196 kb of human sequence, and a 62-kb human homology arm. Human genomic sequences are dark blue, mouse genomic sequences are red, neo selection cassettes are light blue, hyg selection cassettes are orange, and loxP sites are yellow triangles.

Fig. 2.

Humanization of the mouse Ig κ light chain locus. Representations are not drawn to scale. The general scheme for direct genomic swap is depicted in A. The first three steps (B; steps A, B, and C) result in deletion of all mouse V_H, and J_H gene segments, whereas the subsequent five steps (C) result in the insertion of all human J_K gene segments and all human V_K segments in the proximal repeat and deletion of the final hyg selection cassette. Human genomic sequences are dark blue, mouse genomic sequences are red, neo selection cassettes are light blue, hyg selection cassettes are orange, and loxP sites are yellow triangles.

Fig. 3.

Construction of LC-BACvec. (A) The three steps in the construction of the 3hV_H BACvec. pBelo BAC vector is black, human genomic sequences are dark blue, mouse genomic sequences are red, neo/kan selection cassette is light blue, cmR gene is green, specR gene is dark orange, and loxP site is yellow triangle. PFG analysis (B) of three BACs, B1, B2, and B3, after digestion with Not1. Markers M1, M2, and M3 are low range, mid-range, and lambda ladder PFG markers (New England BioLabs), respectively.