Creation of a mouse with human MHC by Chromosomal- and Developmental-engineering methods

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Genetic and chromosomal engineering and transgenic animal technology have greatly contributed to progress of all fields of life sciences, including medicine, agriculture, and pharmacy. We developed chromosome engineering technique to manipulate a large domain of human chromosomes, by introducing several to several-tens Mb portions of human chromosome into chicken DT40 cells, in which frequencies of homologous recombination are several tens to hundreds times higher than those in mammals cell and therefore human chromosome can be efficiently manipulated. Centomere has important roles in production of artificial chromosome with functions properly regulated (Fukagawa et al., 2001 ; Nishihashi et al, 2002).The human MHC locus (4 Mb) consists of three functional domains (classes I, II, and III). We found individual domains to replicate in different periods during S phase and transition of replication timing to occurs at boundaries of the functional domains. French group reported that in the … More case of the pig MHC locus, centromere exists in the boundary of the classes II and III. In this boundary in the human MHC locus we found characteristic sequences that have potentiality to form triple helix (Kanaya et al.,). We also found the triplex structure, which is predicted to modulate replication timing, was formed also within the nucleus and specially associated with centromere sequences (Ohno et al., 2002). Centromeric DNA that exists on the artificial chromosome was found in the late period during S phase, and the replication timing changed after integrating into a host's chromosome. To get artificial chromosomes with proper functions accurately regulated, it is important to clarify the centromeric structures and functions. We analyzed centromeric proteins using gene knocking-out method developed in chicken DT40 and also measured replication timing of the human 6th chromosome with MHC locus using DNA chip techniques. Then, we attempted to transfer human chromosomes from DT40 cells to mouse ES cells.The knowledge acquired in the present study has given basic knowledge to create transgenic mice that hold the long functional genome regions such as human MHC locus. Less