GENOMICS 35, 405–414 (1996) ARTICLE NO. 0379 Antibody Expression from the Core Region of the Human IgH Locus Reconstructed in Transgenic Mice Using Bacteriophage P1 Clones S IMON D. WAGNER ,GIDEON GROSS , 1 GRAHAM P. C OOK, S ARAH L. DAVIES , AND M ICHAEL S. NEUBERGER 2 MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom Received February 14, 1996; accepted May 7, 1996 et al., 1994a,b). Mouse lines that carry transgenic hu- Mice carrying transgenic human immunoglobulin man immunoglobulin gene miniloci have been created; gene miniloci can be used for the production of human the immunoglobulin V, D, and J segments in these monoclonal antibodies. The human variable region (V) miniloci undergo productive rearrangement, yielding a gene segments in these miniloci undergo productive repertoire of lymphocytes making antibodies with hu- rearrangement in mouse lymphoid tissue to yield a man polypeptide chains. Most such mice carry miniloci population of B lymphocytes expressing a repertoire of put together using bacterial plasmid vectors. This obvi- antibodies. Many of the miniloci studied to date have ously limits the size of the miniloci that can be intro- included only a small number of germline gene seg- duced into the mouse germline and may affect both the ments in an artificially compact configuration. Here antibody repertoire that can be produced and its proper we describe the use of the bacteriophage P1 cloning expression. system to create mice carrying the core region of the Given that the human immunoglobulin heavy human immunoglobulin heavy chain (IgH) locus. chain (IgH) locus is 1.5 Mb long (Hofker et al., 1989; Three P1 clones carrying overlapping regions of the Cook et al., 1994), other approaches need to be fol- human IgH locus (spanning the five J H -proximal V H lowed to create near locus-sized transgenes. We and segments, the entire D H and J H clusters, and the Cm others have previously described antibody expression and Cd constant regions) were injected into mouse from human immunoglobulin YACs introduced into eggs and appear to have reconstituted the core region the mouse genome. Thus, YACs were introduced into of the locus (ú180 kb) following homologous recombi- the mouse germline by the embryonic stem (ES) cell nation with each other. While this translocus yielded route, in which the DNA is introduced into the ES a titer of serum immunoglobulin similar to that ob- tained with a smaller plasmid-based minilocus, the cells by lipofection (Choi et al., 1993; Strauss et al., P1-based locus gave rise to substantially greater diver- 1993) or spheroplast fusion (Davies et al., 1993; Jako- sification by somatic hypermutation. Such diversifi- bovits et al., 1993). The transfected ES cells were cation is important for obtaining high-affinity antibod- then used to make blastocyst chimeras, and the chi- ies. The results show the usefulness of the P1 system in meras were bred to obtain transmission through the facilitating the manipulation and recreation of large mouse germline. An alternative, more direct strategy transgenes.  1996 Academic Press, Inc. is the direct microinjection of purified YAC DNA into zygotes (Schedl et al., 1992, 1993), although technical difficulties have been encountered in obtaining puri- INTRODUCTION fied, intact DNA from large YACs in concentrations sufficient for microinjection as well as in avoiding The introduction of large DNA molecules into the subsequent shearing (Gnirke et al., 1993; and our mouse germline forms the basis of a major approach to own experience). the production of human monoclonal antibodies (Bru ¨ g- Here, we have examined the possibility of using gemann et al., 1989, 1991; Bru ¨ ggemann and Neu- bacteriophage P1 clones to reconstitute a core human berger, 1991; Davies et al., 1993; Green et al., 1994; IgH locus in transgenic mice. Phage P1 clones contain Lonberg et al., 1994; Taylor et al., 1992, 1993; Wagner about 100 kb of inserted DNA and are propagated in Escherichia coli (Sternberg, 1990, 1994). DNA from 1 Present address: Migal, Galilee Technological Center, Kiryat these clones can be obtained in good yield and there- Shmona, Israel. fore in a high concentration suitable for direct micro- 2 To whom correspondence should be addressed. Telephone: 44 1223 402245. Fax: 44 1223 412178. injection. DNA from P1 clones is therefore more 405 0888-7543/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved.