Efficient Size-Independent Chromosome Delivery from Yeast to Cultured Cell Lines
Journal Article - Open Access
Synthetic Biology and Bioenergy, J. Craig Venter Institute Rockville United States
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The delivery of large DNA vectors 100 000 bpremains a limiting step in the engineering of mammaliancells and the development of human artificialchromosomes HACs. Yeast is commonly used toassemble genetic constructs in the megabase sizerange, and has previously been used to transfer constructsdirectly into cultured cells. We improved thismethod to efficiently deliver large 1.1 Mb syntheticyeast centromeric plasmids YCps to cultured celllines at rates similar to that of 12 kb YCps. Synchronizingcells in mitosis improved the delivery efficiencyby 10-fold and a statistical design of experimentsapproach was employed to boost the vectordelivery rate by nearly 300-fold from 1250 000 to1840 cells, and subsequently optimize the deliveryprocess for multiple mammalian, avian, and insectcell lines. We adapted this method to rapidly delivera 152 kb herpes simplex virus 1 genome cloned inyeast into mammalian cells to produce infectiousvirus.
- Medicine and Medical Research
- Anatomy and Physiology
- Genetic Engineering and Molecular Biology