International Mammalian Genome Society

The 14th International Mouse Genome Conference (2000)

Table of Contents * Genes, Physical Mapping and Sequencing * Gene Identification * Bioinformatics * Common Resources * RIKEN Session * Functional Genomics * Complex Traits * Mutagenesis * Developmental Genetics and Differentiation * Verne Chapman Memorial Lecture I and II

F2. Engineering of Human Chromosomes; Construction of Human Artificial Chromosomes Carrying Over Megabase-Sized Chromosome Inserts

Yoshimi Kuroiwa¹, Kazuma Tomizuka¹, Tokuyuki Shinohara², Yasuhiro Kazuki², Hitoshi Yoshida¹, Mitsuo Oshimura² & Isao Ishida^1
1Pharmaceutical research laboratory, KIRIN BREWERY. CO., LTD., 3 Miyahara-cho Takasaki-shi Gunma 370-1295, Japan. ²Department of Molecular and Cell Genetics, School of Life Sciences, Faculty of Medicine, Tottori University, Nishimachi 86 Yonago, Tottori 683-8503, Japan.

So far, bacterial artificial chromosomes (BACs), P1 phage-derived artificial chromosomes (PACs) and yeast artificial chromosomes (YACs) have been used for cloning of large genomic DNA. Especially, YACs have a cloning capacity of 1-2 megabase (Mb), but such large genomic inserts are sometimes instable and chimeric. Recently, mammalian artificial chromosomes (MACs) have been developed by top-down and bottom-up approaches for stable introduction of large genomic DNA into cells or animals. However, there has been no report about MACs carrying over Mb-sized genomic DNA inserts.

On the other hand, human chromosomes (hChrs) or their fragments themselves have been used for introduction of large human genomic DNA into mice. However, it was reported that mitotic stability of hChrs in mice was different between hChrs. Thus, it was not generally easy to stably maintain any type of hChr for functional analyses in mice. Furthermore, it was difficult to introduce only defined region on hChrs into mice because the fragmentation of hChrs occurred randomly and accidentally.

For stable introduction of over Mb-sized defined region of human chromosomes into cells or animals, we have developed a chromosome-cloning system where only defined human chromosome region was cloned into a stable human mini-chromosome vector by combination of Cre/loxP-mediated chromosome translocation with telomere-directed chromosome truncation in homologous recombination-proficient chicken DT40 cells. By cloning into the stable mini-chromosome vector, the 10 Mb-sized defined region derived from the mitotically instable human chromosome was mitotically stabilized in mouse ES cells as well as the original mini-chromosome vector itself. This human artificial chromosome (HAC) carrying the 10 Mb-sized defined human chromosome insert was also stably maintained in mice. Furthermore, we demonstrate functional expression of human genes on the HAC in mice. This HAC construction system will enable us to clone, stabilize and functionally analyze over Mb-sized defined human chromosome region in vitro and vivo (Nature Biotech., in press).