Abstract
Microcell-mediated chromosome transfer (MMCT) is an essential step for introducing chromosomes from donor cells to recipient cells. MMCT allows not only for genetic/epigenetic analysis of specific chromosomes, but also for utilization of human and mouse artificial chromosomes (HACs/MACs) as gene delivery vectors. Although the scientific demand for genome scale analyses is increasing, the poor transfer efficiency of the current method has hampered the application of chromosome engineering technology. Here, we developed a highly efficient chromosome transfer method, called retro-MMCT, which is based on Chinese hamster ovary cells expressing envelope proteins derived from ecotropic or amphotropic murine leukemia viruses. Using this method, we transferred MACs to NIH3T3 cells with 26.5 times greater efficiency than that obtained using the conventional MMCT method. Retro-MMCT was applicable to a variety of recipient cells, including embryonic stem cells. Moreover, retro-MMCT enabled efficient transfer of MAC to recipient cells derived from humans, monkeys, mice, rats, and rabbits. These results demonstrate the utility of retro-MMCT for the efficient transfer of chromosomes to various types of target cell.
Highlights
Human artificial chromosomes (HACs) and mouse artificial chromosomes (MACs) are unique gene delivery vectors that behave like natural chromosomes in mammalian host cells [1, 2]
The low efficiency of the conventional Microcell-mediated chromosome transfer (MMCT) method is an obstacle to the advancement of chromosome engineering technology
We overcame this limitation by developing the retro-MMCT method, which enables the introduction of MAC1 to NIH3T3 cells at a frequency of 5.5 × 10−3 colonies per recipient cell without any manipulation of the recipient cells
Summary
Human artificial chromosomes (HACs) and mouse artificial chromosomes (MACs) are unique gene delivery vectors that behave like natural chromosomes in mammalian host cells [1, 2]. Ecotropic MLV recognizes only mouse and rat cells by binding to cationic amino acid transporter-1 (Cat-1), whereas amphotropic MLV infects a wide range of mammalian cells (including mouse, rat, rabbit, monkey, and human) by binding to the sodium-dependent phosphate transporter, Pit-2. Both receptors are ubiquitously expressed membrane proteins conserved in mammals. CHO cells are completely resistant to infection by ecotropic and amphotropic MLVs [19] Based on these properties, we decided to utilize the R-peptide-deleted Env protein of MLVs as the fusogen for donor CHO-derived microcells to develop a highly efficient MMCT method
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