Human Cell Line Panel With Human/Mouse Artificial Chromosomes for Functional Analyses of Desired Genes

Abstract Human artificial chromosomes (HACs) and mouse artificial chromosomes (MACs) are non-integrating chromosomal gene delivery vectors for molecular biology research. Recently, microcell-mediated chromosome transfer of HACs/MACs has been achieved into various human cells including human immortalised mesenchymal stem cells (hiMSCs) and human induced pluripotent stem cells (hiPSCs). However, the conventional strategy of gene-introduction with HAC/MAC required laborious and time-consuming stepwise isolation of clones for gene loading into HACs/MACs in donor cell lines (CHO and A9) and then transferring the HAC/MAC into cells via microcell-mediated chromosome transfer (MMCT). To overcome these limitations and accelerate chromosome vector based functional assay in human cells, we established various human cell lines (HEK293, HT1080, hiMSCs, and hiPSCs) with HACs/MACs that harbour a gene-loading site via MMCT. Model genes, such as tdTomato, TagBFP2, and ELuc, were introduced into the premade HAC/MAC-introduced cell lines via the Cre-loxP system or simultaneous insertion of multiple gene-loading vectors (SIM system). The model genes on the HACs/MACs were stably expressed and the HACs/MACs were stably maintained in the cell lines. Thus, our strategy using the HAC/MAC-containing cell line panel has dramatically simplified and accelerated gene introduction via HACs/MACs, thereby facilitating functional analyses of introduced genes.