Microcell-mediated chromosome transfer (MMCT) is a technique to transfer a chromosome from defined donor cells into recipient cells and to manipulate chromosomes as gene delivery vectors and open a new avenue in somatic cell genetics. However, it is difficult to uncover the function of a single specific gene via the transfer of an entire chromosome or fragment, because each chromosome or fragment contains a set of numerous genes. Thus, alternative tools are human artificial chromosome (HAC) and mouse artificial chromosome (MAC) vectors, which can carry a gene or genes of interest. HACs/MACs have been generated mainly by either a "top-down approach" (engineered creation) or a "bottom-up approach" (de novo creation). HACs/MACs with one or more acceptor sites exhibit several characteristics required by an ideal gene delivery vector, including stable episomal maintenance and the capacity to carry large genomic loci plus their regulatory elements, thus allowing the physiological regulation of the introduced gene in a manner similar to that of native chromosomes. The MMCT technique is also applied for manipulating HACs and MACs in donor cells and delivering them to recipient cells. This review describes the lessons learned and prospects identified from studies on the construction of HACs and MACs, and their ability to drive exogenous gene expression in cultured cells and transgenic animals via MMCT. New avenues for a variety of applications to bio-medical challenges are also proposed.