Abstract
Human artificial chromosomes (HACs) are important tools for epigenetic engineering, for measuring chromosome instability (CIN), and for possible gene therapy. However, their use in the latter is potentially limited because the input HAC-seeding DNA can undergo an unpredictable series of rearrangements during HAC formation. As a result, after transfection and HAC formation, each cell clone contains a HAC with a unique structure that cannot be precisely predicted from the structure of the HAC-seeding DNA. Although it has been reported that these rearrangements can happen, the timing and mechanism of their formation has yet to be described. Here we synthesized a HAC-seeding DNA with two distinct structural domains and introduced it into HT1080 cells. We characterized a number of HAC-containing clones and subclones to track DNA rearrangements during HAC establishment. We demonstrated that rearrangements can occur early during HAC formation. Subsequently, the established HAC genomic organization is stably maintained across many cell generations. Thus, early stages in HAC formation appear to at least occasionally involve a process of DNA shredding and shuffling that resembles chromothripsis, an important hallmark of many cancer types. Understanding these events during HAC formation has critical implications for future efforts aimed at synthesizing and exploiting synthetic human chromosomes.
Highlights
Human artificial chromosomes (HACs) are important tools for epigenetic engineering, for measuring chromosome instability (CIN), and for possible gene therapy
Containing centrochromatin array was designed to resemble the previously published alphoidtetO HAC,[2] but in this case, using 11-mer (1886 bp) high order repeats (HORs) of alphoid type I DNA from the centromere in human chromosome 21. Each monomer of this synthetic HOR contains either a 17 bp CENP-B box, essential for CENP-A deposition,[29,33] or a 39 bp tetracycline operator (TetO) targetable sequence, which is the binding site for E. coli tetracycline repressor (TetR). This dimer is the basic unit for the so-called α21-ITetO (TetO) array, which consists of alternating CENP-B-containing and Tet operator (TetO)
The efficiency of alphoid2domain HAC formation in HT1080 was higher than that typically seen with other HACs.[2,4]
Summary
Human artificial chromosomes (HACs) are important tools for epigenetic engineering, for measuring chromosome instability (CIN), and for possible gene therapy Their use in the latter is potentially limited because the input HACseeding DNA can undergo an unpredictable series of rearrangements during HAC formation. That analysis found complex rearrangements in the organization of its seeding DNA during this multimerization process, including inversions and deletions.[3] These rearrangements are unpredictable and uncontrollable as they occur during the clonal expansion before HAC-bearing cell lines are established and identified. This is the time during which the HAC-seeding DNA is forming a functional centromere, an absolute. Control and original DNA as in C. (E) CHEF analysis of bacterial colony #1 DNA (in duplicate) digested with NotI and BamHI to release the synthetic DNA (m and M, markers)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
