Partial Genome Transfer in Mammals

In mammals, a method for partial transfer called chromosome-mediated gene transfer (CMGT) was established more than three decades ago, which allowed the transfer of subchromosome fragments from Chinese hamster to mouse (McBride and Ozer, 1973). The method consists of isolation of metaphase chromosomes from cells arrested in mitosis by colchicines or col- cemid. The donor chromosome enters recipient cells by phagocytosis, which is facilitated by the addition of poly-L- ornithine. The donor genotype carries com- plementary genes that, when transferred to the recipient cells, allow their proliferation in a selective medium. Isozyme analysis of transformant cell lines demonstrate expres- sion of the complementary gene derived from the donor cells (McBride and Ozer, 1973). This method was later improved by Miller and Ruddle (1978) to increase the frequency of donor chromosome transfer. Since CMGT transfers chromosome frag- ments, the breakage event disrupts syntenic relationships of genes on a chromosome and, therefore, this method can be used to produce mapping data (Klobutcher and Ruddle, 1981). CMGT mapping was suc- cessfully used to map genes in human chro- mosome 17 (Klobutcher and Ruddle, 1979). The CMGT procedure may be stable or unstable, and no donor chromosome frag- ments can be detected cytologically (McBride and Ozer, 1973) even though the transfer is confi rmed by gene complemen- tarity.

Another technique called microcell- mediated chromosome transfer was later developed by Fournier and Ruddle (1977), which enabled them to transfer 1–5 murine

chromosomes to mouse, hamster or human recipient cells. Unlike CMGT, microcell- mediated chromosome transfer allows intact chromosomes to be transferred from one cell to another, resulting in karyotypi- cally simple somatic cell hybrids for cyto- genetic and genetic analysis (Killary and Lott, 1996). To date, microcell-mediated chromosome transfer is one of the most important tools in mammalian cells and especially for humans for gene mapping, analysis of gene function and molecular cloning of defi ned chromosomal regions (Jacob et al., 1999). Most of the 22 human chromosomes are currently available in monochromosomal cell hybrids, produced using microcell-mediated chromosome transfer (Cuthbert et al., 1995; Murakami et al., 2000; Inoue et al., 2001). The proce- dure, as initially set up (Fournier and Ruddle, 1977), consists of exposure of donor cells to colcemid, a microtubule polymerization inhibitor, which prevents formation of a functional mitotic spindle, and thereby arrests the cells in metaphase. This treatment will induce chromosomes from donor cells to be scattered throughout the cytoplasm. When the cells eventually exit mitosis, a nuclear membrane reforms around a single chromosome or small clus- ters of chromosomes to produce micronu- clei. Individual micronuclei may be isolated from the micronucleated cells by the action of cytochalasin B (CB) together with gradient centrifugation to form micro- cells. CB induces nuclear extrusion which, when combined with centrifugation, causes extruded micronuclei to break out on a thin rind of cytoplasm (McNeill and Brown, 1980). Purer microcells can be pro- duced by incorporating a microfi ltration procedure after the centrifugation step (Stubblefi eld and Pershouse, 1992). The microcells are then fused with recipient cells to produce microcell hybrids.