Human chromosomes or chromosome fragments derived from normal fibroblasts were introduced into mouse embryonic stem (ES) cells via microcell-mediated chromosome transfer (MMCT) and viable chimaeric mice were produced from them. Transferred chromosomes were stably retained, and human genes, including immunoglobulin (Ig) kappa, heavy, lambda genes, were expressed in proper tissue-specific manner in adult chimaeric tissues. In the case of a human chromosome (hChr.) 2-derived fragment, it was found to be transmitted to the offspring through the germline. Our study demonstrates that MMCT allows for introduction of very large amounts of foreign genetic material into mice. This novel procedure will facilitate the functional analyses of human genomes in vivo.

The use of a human chromosome or its fragment as a vector for animal transgenesis may facilitate functional studies of large human genomic regions. We describe here the generation and analysis of double trans-chromosomic (Tc) mice harboring two individual human chromosome fragments (hCFs). Two transmittable hCFs, one containing the Ig heavy chain locus (IgH, Ϸ1.5 Mb) and the other the light chain locus (Ig, Ϸ2 Mb), were introduced into a mouse strain whose endogenous IgH and Ig loci were inactivated. In the resultant double-Tc͞double-knockout mice, substantial proportion of the somatic cells retained both hCFs, and the rescue in the defect of Ig production was shown by high level expression of human Ig heavy and chains in the absence of mouse heavy and chains. In addition, serum expression profiles of four human Ig ␥ subclasses resembled those seen in humans. They mounted an antigen-specific human antibody response upon immunization with human serum albumin, and human serum albumin-specific human monoclonal antibodies with various isotypes were obtained from them. These results represent a generation of mice with ''humanized'' loci by using the transmittable hCFs, which suggest that the Tc technology may allow for the humanization of over megabase-sized, complex loci in mice or other animals. Such animals may be useful not only for studying in vivo functions of the human genome but also for obtaining various therapeutic products.

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Along with the T-cell receptor and immunoglobulin, the major histocompatibility complex (MHC) plays a key role in mounting immune responses to foreign antigen. To gain insights into the evolution of the MHC, class II A cDNA clones were isolated from nurse sharks, a member of the class of cartilaginous fish. Two closely related cDNA clones, which might encode allelic products, were identified; of the three amino acid substitutions found in the al domain, two were located at positions postulated to interact with processed peptides. The deduced nurse shark MHC class H a chains showed conspicuous structural similarity to their mammalian counterparts. Isolation of cDNA clones encoding typical MHC class II a chains was unexpected since no direct evidence for T-cellmediated immune responses has been obtained in the cartilaginous fish. The cartilaginous fish is phylogenetically the most primitive class of vertebrates from which any MHC gene has been isolated.Genes of the major histocompatibility complex (MHC) encode two classes of structurally similar, but functionally distinct, glycoproteins that present peptides to T cells (1, 2). In general, MHC class I molecules, consisting of an a chain and ,82-microglobulin, present peptides derived from endogenously synthesized proteins to CD8+ T cells. The peptides are bound by two membrane-distal domains (al and a2), which form a deep cleft made up of an eight-stranded (3-pleated sheet topped by two long a-helices (3, 4). MHC class II molecules are heterodimers that, as a rule, present peptides derived from exogenously acquired proteins to CD4+ T cells. The peptides are bound by two membranedistal domains (al and (31), assumed to form a cleft structurally similar to that of MHC class I molecules (5).At which stage of evolution did an ancestral MHC molecule emerge, and when and how did it diversify into two classes of functionally specialized molecules? Was an ancestral MHC molecule class I-like or class II-like? These questions, which are of fundamental importance in understanding the evolution of immunity (6-8), can be addressed only by studying MHC genes of primitive creatures. In addition, the structure of such MHC genes might provide a clue to the origin of membrane-distal, peptide-binding domains (9) and to the primordial function of MHC molecules. With these points in mind, we have embarked upon a project aimed at isolating MHC genes from primitive vertebrates (10).t