LAUSR

Abstract The delivery of large DNA vectors (>100 000 bp) remains a limiting step in the engineering of mammalian cells and the development of human artificial chromosomes (HACs). Yeast is commonly used to assemble genetic constructs in the megabase size range, and has previously been used to transfer constructs directly into cultured cells. We improved this method to efficiently deliver large (1.1 Mb) synthetic yeast centromeric plasmids (YCps) to cultured cell lines at rates similar to that of 12 kb YCps. Synchronizing cells in mitosis improved the delivery efficiency by 10-fold and a statistical design of experiments approach was employed to boost the vector delivery rate by nearly 300-fold from 1/250 000 to 1/840 cells, and subsequently optimize the delivery process for multiple mammalian, avian, and insect cell lines. We adapted this method to rapidly deliver a 152 kb herpes simplex virus 1 genome cloned in yeast into mammalian cells to produce infectious virus.