Abstract
Human artificial chromosomes (HACs) are gene-delivery vectors suitable for introducing large DNA fragments into mammalian cells. Although a HAC theoretically incorporates multiple gene expression cassettes of unlimited DNA size, its application has been limited because the conventional gene-loading system accepts only one gene-loading vector (GLV) into a HAC. We report a novel method for the simultaneous or sequential integration of multiple GLVs into a HAC vector (designated as the SIM system) via combined usage of Cre, FLP, Bxb1, and φC31 recombinase/integrase. As a proof of principle, we first attempted simultaneous integration of three GLVs encoding EGFP, Venus, and TdTomato into a gene-loading site of a HAC in CHO cells. These cells successfully expressed all three fluorescent proteins. Furthermore, microcell-mediated transfer of HACs enabled the expression of those fluorescent proteins in recipient cells. We next demonstrated that GLVs could be introduced into a HAC one-by-one via reciprocal usage of recombinase/integrase. Lastly, we introduced a fourth GLV into a HAC after simultaneous integration of three GLVs by FLP-mediated DNA recombination. The SIM system expands the applicability of HAC vectors and is useful for various biomedical studies, including cell reprogramming.
Highlights
Human artificial chromosomes (HACs) and mouse artificial chromosomes (MACs) are chromosomal gene-delivery vectors
Construction of gene-loading vector (GLV) for the SIM system We first constructed a variety of modules called SIM cassettes, which consist of recognition sequences of Cre, FLP, Bxb1, and/or QC31 recombinase/integrase [21], a splicing acceptor sequence and/or drug resistance gene (Fig. 1a, Fig. S1)
We reported the simultaneous integration of up to three GLVs to a gene-loading site of a HAC via the SIM system
Summary
Human artificial chromosomes (HACs) and mouse artificial chromosomes (MACs) are chromosomal gene-delivery vectors. They behave as independent extra chromosomes and are stably maintained in host cells. Since a HAC has no size limitation for insert DNA, it is possible to adjust expression levels of exogenous genes by changing the copy number of their expression units. Another advantage of the HAC expression system is that foreign genes on HACs can be removed by eliminating the HAC itself before therapeutic application [6,8,9,10,11]. HAC/MAC vectors would be useful for such direct reprogramming owing to their superior geneloading capacity
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