Method to Assemble Genomic DNA Fragments or Geneson Human Artificial Chromosome with Regulated Kinetochore Using aMulti-Integrase System

摘要

The production of cells capable of carrying multiple transgenes to Mb-size genomic loci has multiple applications in biomedicine and biotechnology. In order to achieve this goal, three key steps are required: (i) cloning of large genomic segments; (ii) insertion of multiple DNA blocks at a precise location and (iii) the capability to eliminate the assembled region from cells. In this study, we designed the iterative integration system (IIS) that utilizes recombinases Cre, ΦC31 and ΦBT1, and combined it with a human artificial chromosome (HAC) possessing a regulated kinetochore (alphoid^tetO-HAC). We have demonstrated that the IIS-alphoid^tetO-HAC system is a valuable genetic tool by reassembling a functional gene from multiple segments on the HAC. IIS-alphoid^tetO-HAC has several notable advantages over other artificial chromosome-based systems. This includes the potential to assemble an unlimited number of genomic DNA segments; a DNA assembly process that leaves only a small insertion (<60 bp) scar between adjacent DNA, allowing genes reassembled from segments to be spliced correctly; a marker exchange system that also changes cell color, and counter-selection markersat each DNA insertion step, simplifying selection of correct clones;and presence of an error proofing mechanism to remove cells with misincorporatedDNA segments, which improves the integrity of assembly. In addition,the IIS-alphoid^tetO-HAC carrying a locus of interest isremovable, offering the unique possibility to revert the cell lineto its pretransformed state and compare the phenotypes of human cellswith and without a functional copy of a gene(s). Thus, IIS-alphoid^tetO-HAC allows investigation of complex biomedical pathways,gene(s) regulation, and has the potential to engineer synthetic chromosomeswith a predetermined set of genes.