Generation of a Synthetic Human Chromosome with Two Centromeric Domains for Advanced Epigenetic Engineering Studies

Pesenti, Elisa; Kouprina, Natalay; Liskovykh, Mikhail; Aurich-Costa, Joan; Larionov, Vladimir; Masumoto, Hiroshi; Earnshaw, William C.; Molina, Òscar

doi:10.1021/acssynbio.8b00018

Cited by 16 publications

(45 citation statements)

References 53 publications

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“…At day 0 and 15 after blasticidin withdrawal, the rate of EGFP-positive cells was assessed by flow cytometry, and the rate of HAC loss on the metaphase spreads was counted manually under microscopy 30 metaphase spreads were examined per each cell line. The daily loss rate of the HAC was calculated as previously described [36,37] applying the formula 15 where N 15 is the percent of metaphase spreads containing the HAC or EGFP-positive (for FACS assaying) cells after 15 days of blasticidin withdrawal, N 0 is the percent of metaphase spreads containing the HAC or EGFP-positive cells at day 0 of the blasticidin withdrawal, and R is the daily loss of the HAC.…”

Section: Analysis Of Mitotic Stability Of the Fviii-alphoid Teto -Hacmentioning

confidence: 99%

Human AlphoidtetO Artificial Chromosome as a Gene Therapy Vector for the Developing Hemophilia A Model in Mice

Ponomartsev

Sinenko

Skvortsova

et al. 2020

Cells

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Human artificial chromosomes (HACs), including the de novo synthesized alphoidtetO-HAC, are a powerful tool for introducing genes of interest into eukaryotic cells. HACs are mitotically stable, non-integrative episomal units that have a large transgene insertion capacity and allow efficient and stable transgene expression. Previously, we have shown that the alphoidtetO-HAC vector does not interfere with the pluripotent state and provides stable transgene expression in human induced pluripotent cells (iPSCs) and mouse embryonic stem cells (ESCs). In this study, we have elaborated on a mouse model of ex vivo iPSC- and HAC-based treatment of hemophilia A monogenic disease. iPSCs were developed from FVIIIY/− mutant mice fibroblasts and FVIII cDNA, driven by a ubiquitous promoter, was introduced into the alphoidtetO-HAC in hamster CHO cells. Subsequently, the therapeutic alphoidtetO-HAC-FVIII was transferred into the FVIIIY/– iPSCs via the retro-microcell-mediated chromosome transfer method. The therapeutic HAC was maintained as an episomal non-integrative vector in the mouse iPSCs, showing a constitutive FVIII expression. This study is the first step towards treatment development for hemophilia A monogenic disease with the use of a new generation of the synthetic chromosome vector—the alphoidtetO-HAC.

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Section: Analysis Of Mitotic Stability Of the Fviii-alphoid Teto -Hacmentioning

confidence: 99%

Human AlphoidtetO Artificial Chromosome as a Gene Therapy Vector for the Developing Hemophilia A Model in Mice

Ponomartsev

Sinenko

Skvortsova

et al. 2020

Cells

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“…The alphoid 2domain HAC is formed by two arrays, similarly to a previously constructed alphoid hybrid HAC 4 , but using a ~2.5x larger (~120 kb) HAC-seeding construct. The CENP-B-containing centrochromatin array was designed to resemble the previously published alphoid tetO 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.…”

Section: Array Amplificationmentioning

confidence: 99%

“…Human Artificial Chromosomes (HACs) are non-essential mini-chromosomes that replicate and segregate correctly in human cells 1 . HACs made using synthetic centromeric DNA have been extensively characterized and improved over the last decade [2][3][4][5] . They now represent an important tool to study epigenetic regulation of centromere structure and function 2,[6][7][8][9][10] , to study full-length gene functions in mutant animal or human cells 5,[11][12][13][14][15][16] , to measure chromosome instability (CIN) and identify new targets for cancer therapy [17][18][19] .…”

Section: Introductionmentioning

confidence: 99%

“…Synthetic HACs were originally designed using a "bottom up" approach to contain only pre-defined DNA arrays 1,20,21 . Their design allows the HAC centromeres to be easily modified and inactivated/removed by targeting with chimeric proteins specifically directed to the synthetic DNA 2,4,11,14 . However, they also present a number of challenges that must be overcome to enable them to be exploited fully.…”

Section: Introductionmentioning

confidence: 99%

“…Here, we have performed the first systematic study of rearrangements that occur during HAC formation and determined how they alter the epigenetic landscape in the HAC centromere and how they impact HAC segregation in mitosis. The seeding DNA of the new alphoid 2domain HAC resembles that used to construct the alphoid hybrid HAC described earlier 4 but was much larger, as we hypothesized that this might minimize the need for rearrangements and amplification during HAC formation. The alphoid 2domain…”

Section: Introductionmentioning

confidence: 99%

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Analysis of Complex DNA Rearrangements During Early Stages of HAC Formation

Pesenti

Liskovykh

Okazaki

et al. 2020

Preprint

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AbstractHuman Artificial Chromosomes (HACs) are important tools for epigenetic engineering, for measuring chromosome instability (CIN) and 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.

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Highly Efficient Microcell‐Mediated Transfer of HACs Containing a Genomic Region of Interest into Mammalian Cells

2021

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Human artificial chromosomes (HACs) are considered promising tools for gene delivery, functional analyses, and gene therapy. HACs have the potential to overcome many of the problems caused by the use of viral-based gene transfer systems, such as limited cloning capacity, lack of copy number control, and insertional mutagenesis during integration into host chromosomes. The recently developed alphoid tetO -HAC has an advantage over other HAC vectors because it can be easily eliminated from dividing cells by inactivation of its conditional kinetochore. This provides a unique control mechanism to study phenotypes induced by a gene or genes carried on the HAC. The alphoid tetO -HAC has a single gene acceptor loxP site that allows insertion of an individual gene of interest or a cluster of genes of up to several Mb in size in Chinese hamster ovary (CHO) hybrid cells. The HACs carrying chromosomal copies of genes can then be transferred from these donor CHO cells to different recipient cells of interest via microcell-mediated chromosome transfer (MMCT). Here, we describe a detailed protocol for loading a gene of interest into the alphoid tetO -HAC vector and for the subsequent transfer of the HAC to recipient cells using an improved MMCT protocol. The original MMCT protocol includes treatment of donor cells with colcemid to induce micronucleation, wherein the HAC becomes surrounded with a nuclear membrane. That step is followed by disarrangement of the actin cytoskeleton using cytochalasin B to help induce microcell formation. The updated MMCT protocol, described here, features the replacement of colcemid and cytochalasin B with TN16 + griseofulvin and latrunculin B, respectively, and the use of collagen/laminin surface coating to promote attachment of metaphase cells to plates during micronuclei induction. These modifications increase the efficiency of HAC transfer to recipient cells ten fold. The improved MMCT protocol has been successfully tested on several recipient cell lines, including human mesenchymal stem cells and mouse embryonic stem cells.

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Generation of a Synthetic Human Chromosome with Two Centromeric Domains for Advanced Epigenetic Engineering Studies

Cited by 16 publications

References 53 publications

Human AlphoidtetO Artificial Chromosome as a Gene Therapy Vector for the Developing Hemophilia A Model in Mice

Human AlphoidtetO Artificial Chromosome as a Gene Therapy Vector for the Developing Hemophilia A Model in Mice

Analysis of Complex DNA Rearrangements During Early Stages of HAC Formation

Highly Efficient Microcell‐Mediated Transfer of HACs Containing a Genomic Region of Interest into Mammalian Cells

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