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 (MMCT) of HACs/MACs has been achieved in various human cells that include human immortalised mesenchymal stem cells (hiMSCs) and human induced pluripotent stem cells (hiPSCs). However, the conventional strategy of gene introduction with HACs/MACs requires 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 MMCT. To overcome these limitations and accelerate chromosome vector-based functional assays 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 these preprepared HAC/MAC-introduced cell lines via the Cre-loxP system or simultaneous insertion of multiple gene-loading vectors. 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 this HAC/MAC-containing cell line panel has dramatically simplified and accelerated gene introduction via HACs/MACs.
Highlights
Human artificial chromosomes (HACs) and mouse artificial chromosomes (MACs) are non-integrating chromosomal gene delivery vectors for molecular biology research
HACs were derived from human chromosome 21, such as 21HAC1 without EGFP and 21HAC2 with EGFP41, and MACs were derived from mouse chromosome 11, such as MAC2 without EGFP, MAC4 with EGFP 37,42,43, and MAC6 with EGFP
The results showed that one HAC/MAC was maintained in each cell and that the transfected plasmid was inserted into the HAC/MAC as expected (Supplementary Fig. S3c,d)
Summary
Human artificial chromosomes (HACs) and mouse artificial chromosomes (MACs) are non-integrating chromosomal gene delivery vectors for molecular biology research. The conventional strategy of gene introduction with HACs/MACs requires laborious and time-consuming stepwise isolation of clones for gene loading into HACs/MACs in donor cell lines (CHO and A9) and transferring the HAC/MAC into cells via MMCT To overcome these limitations and accelerate chromosome vector-based functional assays in human cells, we established various human cell lines (HEK293, HT1080, hiMSCs, and hiPSCs) with HACs/MACs that harbour a geneloading site via MMCT. Our strategy using this HAC/MACcontaining cell line panel has dramatically simplified and accelerated gene introduction via HACs/ MACs. Abbreviations HAC Human artificial chromosome MAC Mouse artificial chromosome MMCT Microcell-mediated chromosome transfer hiMSC Human immortalised mesenchymal stem cell hiPSC Human induced pluripotent stem cell Tc Trans-chromosomic SIM Simultaneous or sequential insertion of multiple genes-loading vectors HPRT Hypoxanthine-guanine phosphoribosyltransferase MI Multiple integrase CRISPR Clustered regularly interspaced short palindromic repeats Cas[9] CRISPR-associated protein 9 MV Measles virus. We further attempted to adapt the Cre-loxP system (Supplementary Fig. S2a) and SIM system for multiple gene loadings (Supplementary Fig. S2b) in the generated human cell lines with HACs/MACs
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