human artificial chromosome HAC

CENP-B binds to CENP-B boxes on centromeric satellite DNAs (known as alphoid DNA in humans). CENP-B maintains kinetochore function through interactions with CENP-A nucleosomes and CENP-C. CENP-B binding to transfected alphoid DNA can induce de novo CENP-A assembly, functional centromere and kinetochore formation, and subsequent human artificial chromosome (HAC) formation. Furthermore, CENP-B also facilitates H3K9 (histone H3 lysine 9) trimethylation on alphoid DNA, mediated by Suv39h1, at ectopic alphoid DNA integration sites...

Most eukaryotic centromeres are located within heterochromatic regions. Paradoxically, heterochromatin can also antagonize de novo centromere formation and some centromeres lack it altogether. In order to investigate the importance of heterochromatin at centromeres, we used epigenetic engineering of a synthetic alphoidtetO Human Artificial Chromosome (HAC), to which chimeric proteins can be targeted. By tethering the JMJD2D demethylase, we removed heterochromatin mark H3K9me3 specifically from the HAC centromere...

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)...

Centromeres are essential components of all eukaryotic chromosomes, including artificial/synthetic ones built in the laboratory. In humans, centromeres are typically located on repetitive α-satellite DNA, and these sequences are the "major ingredient" in first-generation human artificial chromosomes (HACs). Repetitive centromeric sequences present a major challenge for the design of synthetic mammalian chromosomes because they are difficult to synthesize, assemble, and characterize. Additionally, in most eukaryotes, centromeres are defined epigenetically...

The formation of de novo centromeres on artificial chromosomes in humans (HACs) and fission yeast (SpYACs) has provided much insights to the epigenetic and genetic control on regional centromere establishment and maintenance. Similarly, the use of artificial chromosomes in point centromeric budding yeast Saccharomyces cerevisiae (ScYACs) and holocentric Caenorhabditis elegans (WACs) has revealed epigenetic regulation in the originally thought purely genetically-determined point centromeres and some centromeric DNA sequence features in holocentromeres, respectively...

After the construction of genomic libraries with yeast artificial chromosomes in the late 1980's for gene isolation and expression studies in cells, human artificial chromosomes were then a natural development in the 1990's, based on the same principles of formation requiring centromeric sequences for generating functional artificial chromosomes. Over the past twenty years, they became a useful research tool for understanding human chromosome structure and organization, and important vectors for expression of large genes and gene loci and the regulatory regions for full expression...

Chromosomes and chromosomal gene delivery vectors, human/mouse artificial chromosomes (HACs/MACs), can introduce megabase-order DNA sequences into target cells and are used for applications including gene mapping, gene expression control, gene/cell therapy, and the development of humanized animals and animal models of human disease. Microcell-mediated chromosome transfer (MMCT), which enables chromosome transfer from donor cells to target cells, is a key technology for these applications. In this review, we summarize the principles of gene transfer with HACs/MACs; their engineering, characteristics, and utility; and recent advances in the chromosome transfer technology...

The centromere is a specialized chromosomal locus required for accurate chromosome segregation. Heterochromatin also assembles around centromere chromatin and forms a base that supports sister chromatid cohesion until anaphase begins. Both centromere chromatin and heterochromatin assemble on a centromeric DNA sequence, a highly repetitive sequence called alphoid DNA (α-satellite DNA) in humans. Alphoid DNA can form a de novo centromere and subsequent human artificial chromosome (HAC) when introduced into the human culture cells HT1080...

The gene therapy approach aiming at curing various human diseases began to develop as a technology from early eighties of the last century. To date the delivery of therapeutic genes are mainly mediated by virus-based, predominantly, non-integrated virus vectors. These gene delivery approaches have several fundamental limitations on the way of efficient deployment in clinical gene therapy. A totally different approach was suggested about 20 years ago when episomal non-integrative artificial chromosome-based vectors featuring large size inserts (even native gene loci) advanced to the stage...

Gene expression studies and gene therapy require efficient gene delivery into cells. Different technologies by viral and non-viral mechanisms have been used for gene delivery into cells. Small gene vectors transfer across the cell membrane with a relatively high efficiency, but not large genes or entire loci spanning several kilobases, which do not remain intact following introduction. Previously, we developed an efficient delivery system based on herpes virus simplex type 1 (HSV-1) amplicons to transfer large fragments of DNA incorporated in human artificial chromosome vectors into the nucleus of human cells...

Chromosomal instability (CIN) is one of the characteristics of cancer inherent for tumor initiation and progression, which is defined as a persistent, high rate of gain/loss of whole chromosomes. In the vast majority of human tumors the molecular basis of CIN remains unknown. The development of a conceptually simple colony color sectoring assay that measures yeast artificial chromosome (YAC) loss provided a powerful genetic tool to assess the rate of chromosome mis-segregation and also identified 937 yeast genes involved in this process...

Chromosome manipulation is a useful technique in biological science. We have constructed human artificial chromosomes (HACs) based on the transfection of centromeric alphoid DNA precursors into cultured human cells. Moreover, HAC-based technology has been developed into a novel gene expression vector tool for introducing large-size genomic DNA. This technique provides natural expression, as well as stable expression without the gene silencing that often occurs with conventional vectors in mammalian cells. Here we review the properties of HACs, and issues regarding the use of HAC technology for basic and applied research...

Human artificial chromosomes (HACs) are a potentially powerful technique for genomic engineering, but their use is limited by the repetitive centromeric alpha-satellite DNA needed to form a centromere. A new study presents a method to induce HAC centromere formation on non-repetitive templates through sequence-directed CENP-A nucleosome seeding.

Gene amplification methods play a crucial role in establishment of cells that produce high levels of recombinant protein. However, the stability of such cell lines and the level of recombinant protein produced continue to be suboptimal. Here, we used a combination of a human artificial chromosome (HAC) vector and initiation region (IR)/matrix attachment region (MAR) gene amplification method to establish stable cells that produce high levels of recombinant protein. Amplification of Enhanced green fluorescent protein (EGFP) was induced on a HAC carrying EGFP gene and IR/MAR sequences (EGFP MAR-HAC) in CHO DG44 cells...

Stem cells offer great hope for the therapy of neurological disorders. Using a human artificial chromosome (HAC), we generated modified mesenchymal stem cells (MSCs), termed HAC-MSC that express 3 growth factors and 2 marker proteins including luciferase, and previously demonstrated that intrathecal administration of HAC-MSCs extended the lifespan in a mouse model of amyotrophic lateral sclerosis (ALS). However, donor cells disappeared rapidly after transplantation. To overcome this poor survival, we transplanted the HAC-MSCs as a sheet structure which retained the extracellular matrix...

One of the hallmarks of cancer is chromosome instability (CIN), which leads to aneuploidy, translocations and other chromosome aberrations. However, in the vast majority of human tumors the molecular basis of CIN remains unknown, partially because not all genes controlling chromosome transmission have yet been identified. To address this question, we have developed an experimental high-throughput imaging (HTI) siRNA assay that allows the identification of novel CIN genes. Our method uses a human artificial chromosome (HAC) expressing the GFP transgene...

Recent breakthroughs with synthetic budding yeast chromosomes expedite the creation of synthetic mammalian chromosomes and genomes. Mammals, unlike budding yeast, depend on the histone H3 variant, CENP-A, to epigenetically specify the location of the centromere-the locus essential for chromosome segregation. Prior human artificial chromosomes (HACs) required large arrays of centromeric α-satellite repeats harboring binding sites for the DNA sequence-specific binding protein, CENP-B. We report the development of a type of HAC that functions independently of these constraints...

Completion of the human genome sequence and recent advances in engineering technologies have enabled an unprecedented level of understanding of DNA variations and their contribution to human diseases and cellular functions. However, in some cases, long-read sequencing technologies do not allow determination of the genomic region carrying a specific mutation (e.g., a mutation located in large segmental duplications). Transformation-associated recombination (TAR) cloning allows selective, most accurate, efficient, and rapid isolation of a given genomic fragment or a full-length gene from simple and complex genomes...

To address the unmet needs for human polyclonal antibodies both as therapeutics and diagnostic reagents, building upon our previously established transchromosomic (Tc) cattle platform, we report herein the development of a Tc goat system expressing human polyclonal antibodies in their sera. In the Tc goat system, a human artificial chromosome (HAC) comprising the entire human immunoglobulin (Ig) gene repertoire in the germline configuration was introduced into the genetic makeup of the domestic goat. We achieved this by transferring the HAC into goat fetal fibroblast cells followed by somatic cell nuclear transfer for Tc goat production...

Accurate chromosome segregation is essential for cell proliferation. The centromere is a specialized chromosomal locus, on which the kinetochore structure is formed. The centromere/kinetochore is required for the equal separation of sister chromatids to daughter cells. Here, we review recent findings on centromere-specific chromatin, including its constitutive protein components, its de novo formation and maintenance mechanisms, and our progress in analyses with synthetic human artificial chromosomes (HACs)...