RESUMO
Telomerase is a ribonucleoprotein ribonucleic enzyme that elongates telomere repeat sequences at the ends of chromosomes and contributes to cellular immortalization. The catalytic component of telomerase, human telomerase reverse transcriptase (hTERT), has been observed to be reactivated in immortalized cells. Notably, most cancer cells have been found to have active hTERT mRNA transcription, resulting in continuous cell division, which is crucial for malignant transformation. Therefore, discovering mechanisms underlying the regulation of hTERT transcription is an attractive target for cancer-specific treatments.Loss of heterozygosity (LOH) of chromosome 3p21.3 has been frequently observed in human oral squamous cell carcinoma (OSCC). Moreover, we previously reported that HSC3 OSCC microcell hybrid clones with an introduced human chromosome 3 (HSC3#3) showed inhibition of hTERT transcription compared with the parental HSC3 cells. This study examined whether hTERT transcription regulators are present in the 3p21.3 region. We constructed a human artificial chromosome (HAC) vector (3p21.3-HAC) with only the 3p21.3-p22.2 region and performed functional analysis using the 3p21.3-HAC. HSC3 microcell hybrid clones with an introduced 3p21.3-HAC exhibited significant suppression of hTERT transcription, similar to the microcell hybrid clones with an intact chromosome 3. In contrast, HSC3 clones with truncated chromosome 3 with deletion of the 3p21.3 region (3delp21.3) showed no effect on hTERT expression levels. These results provide direct evidence that hTERT suppressor gene(s) were retained in the 3p21.3 region, suggesting that the presence of regulatory factors that control telomerase enzyme activity may be involved in the development of OSCC.
Assuntos
Carcinoma de Células Escamosas , Cromossomos Artificiais Humanos , Neoplasias Bucais , Telomerase , Humanos , Telomerase/genética , Telomerase/metabolismo , Carcinoma de Células Escamosas/genética , Cromossomos Artificiais Humanos/metabolismo , Neoplasias Bucais/genética , Transcrição GênicaRESUMO
Human artificial chromosomes (HACs) and mouse artificial chromosomes (MACs) are non-integrating chromosomal gene delivery vectors for molecular biology research. Recently, microcell-mediated chromosome transfer (MMCT) of HACs/MACs has been achieved in various human cells that include human immortalised mesenchymal stem cells (hiMSCs) and human induced pluripotent stem cells (hiPSCs). However, the conventional strategy of gene introduction with HACs/MACs requires laborious and time-consuming stepwise isolation of clones for gene loading into HACs/MACs in donor cell lines (CHO and A9) and then transferring the HAC/MAC into cells via MMCT. To overcome these limitations and accelerate chromosome vector-based functional assays in human cells, we established various human cell lines (HEK293, HT1080, hiMSCs, and hiPSCs) with HACs/MACs that harbour a gene-loading site via MMCT. Model genes, such as tdTomato, TagBFP2, and ELuc, were introduced into these preprepared HAC/MAC-introduced cell lines via the Cre-loxP system or simultaneous insertion of multiple gene-loading vectors. The model genes on the HACs/MACs were stably expressed and the HACs/MACs were stably maintained in the cell lines. Thus, our strategy using this HAC/MAC-containing cell line panel has dramatically simplified and accelerated gene introduction via HACs/MACs.
Assuntos
Cromossomos Artificiais Humanos , Técnicas de Transferência de Genes , Animais , Linhagem Celular , Vetores Genéticos , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas , Células-Tronco Mesenquimais , Camundongos , Biologia MolecularRESUMO
A novel approach in gene therapy was introduced 20 years ago since artificial non-integrative chromosome-based vectors containing gene loci size inserts were engineered. To date, different human artificial chromosomes (HAC) were generated with the use of de novo construction or "top-down" engineering approaches. The HAC-based therapeutic approach includes ex vivo gene transferring and correction of pluripotent stem cells (PSCs) or highly proliferative modified stem cells. The current progress in the technology of induced PSCs, integrating with the HAC technology, resulted in a novel platform of stem cell-based tissue replacement therapy for the treatment of genetic disease. Nowadays, the sophisticated and laborious HAC technology has significantly improved and is now closer to clinical studies. In here, we reviewed the achievements in the technology of de novo synthesized HACs for a chromosome transfer for developing gene therapy tissue replacement models of monogenic human diseases.
Assuntos
Cromossomos Artificiais Humanos/genética , Terapia Genética , Células-Tronco Pluripotentes Induzidas/transplante , Transplante de Células-Tronco , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Vetores Genéticos/uso terapêutico , HumanosRESUMO
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.
Assuntos
Cromossomos Artificiais Humanos/metabolismo , Rearranjo Gênico/fisiologia , Linhagem Celular Tumoral , Centrômero/metabolismo , Proteína B de Centrômero/genética , Instabilidade Cromossômica , Epigênese Genética , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , HumanosRESUMO
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. Since then numerous human artificial chromosome (HAC) vectors were developed by both de novo synthesis and top-down engineering technology. This approach so far is limited to ex vivo gene transfer and correction within highly proliferative or reversibly immortalized precursor stem cells or pluripotent stem cells. Recent breakthrough in generation of induced pluripotent stem cells and embryonic stem cell manipulation give the additional pivotal stimuli to integrate it with the HAC technology and to develop thereby novel approaches to replacement therapies of human genetic diseases. The HAC technology is complex and time consuming while nowadays it has significantly advanced and become notably closer to medical applications. In this review we discuss current advancements in the HAC technology, in particular, in terms of improvement of chromosome transfer method and achievements in developing mouse-based gene therapy tissue replacement models for several monogenic human diseases. The main progress has been done in elaboration of top-down type HAC technology in modeling and preclinical studies of gene therapy treatment for Duchenne muscular dystrophy (DMD) disease.
Assuntos
Cromossomos Artificiais Humanos/fisiologia , Terapia Genética/métodos , Células-Tronco Pluripotentes/transplante , Transplante de Células-Tronco/métodos , Células-Tronco Embrionárias/fisiologia , Técnicas de Transferência de Genes , Terapia Genética/efeitos adversos , Terapia Genética/ética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/transplante , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Células-Tronco Pluripotentes/metabolismo , Transplante de Células-Tronco/efeitos adversos , Transplante de Células-Tronco/éticaRESUMO
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. Similarly, a human artificial chromosome (HAC)-based assay has been recently developed and applied to quantify chromosome mis-segregation events in human cells. This assay allowed identification of novel human CIN genes in the library of protein kinases. Among them are PINK1, TRIO, IRAK1, PNCK, and TAOK1. The HAC-based assay may be applied to screen siRNA, shRNA and CRISPR-based libraries to identify the complete spectrum of CIN genes. This will reveal new insights into mechanisms of chromosome segregation and may expedite the development of novel therapeutic strategies to target the CIN phenotype in cancer cells.
Assuntos
Instabilidade Cromossômica/genética , Segregação de Cromossomos/genética , Cromossomos Artificiais Humanos/genética , Transgenes/genética , Humanos , Neoplasias/genética , Proteínas Quinases/genética , RNA Interferente Pequeno/genéticaRESUMO
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. The expression level of EGFP increased approximately 6-fold compared to the original HAC without IR/MAR sequences. Additionally, anti-vascular endothelial growth factor (VEGF) antibody on a HAC (VEGF MAR-HAC) was also amplified by utilization of this IR/MAR-HAC system, and anti-VEGF antibody levels were approximately 2-fold higher compared with levels in control cells without IR/MAR. Furthermore, the expression of anti-VEGF antibody with VEGF MAR-HAC in CHO-K1 cells increased 2.3-fold compared with that of CHO DG44 cells. Taken together, the IR/MAR-HAC system facilitated amplification of a gene of interest on the HAC vector, and could be used to establish a novel cell line that stably produced protein from mammalian cells.
Assuntos
Cromossomos Artificiais Humanos , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Células CHO , Cricetulus , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Regiões de Interação com a Matriz/genética , Técnicas de Amplificação de Ácido Nucleico , Proteínas Recombinantes/metabolismo , Fator A de Crescimento do Endotélio Vascular/imunologiaRESUMO
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, partly because not all genes controlling chromosome transmission have yet been identified. To address this question, we 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. When this assay was applied to screen an siRNA library of protein kinases, we identified PINK1, TRIO, IRAK1, PNCK, and TAOK1 as potential novel genes whose knockdown induces various mitotic abnormalities and results in chromosome loss. The HAC-based assay can be applied for screening different siRNA libraries (cell cycle regulation, DNA damage response, epigenetics, and transcription factors) to identify additional genes involved in CIN. Identification of the complete spectrum of CIN genes will reveal new insights into mechanisms of chromosome segregation and may expedite the development of novel therapeutic strategies to target the CIN phenotype in cancer cells.
Assuntos
Instabilidade Cromossômica/genética , Cromossomos Humanos/genética , Proteínas Quinases/genética , RNA Interferente Pequeno/genética , Aneuploidia , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/genética , Linhagem Celular Tumoral , Cromossomos Artificiais Humanos/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Quinases Associadas a Receptores de Interleucina-1/genética , Mitose/genética , Proteínas Quinases/isolamento & purificação , Proteínas Serina-Treonina Quinases/genética , RNA de Cadeia Dupla/genética , Transgenes , Translocação Genética/genéticaRESUMO
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. Formed by an initial CENP-A nucleosome seeding strategy, a construct lacking repetitive centromeric DNA formed several self-sufficient HACs that showed no uptake of genomic DNA. In contrast to traditional α-satellite HAC formation, the non-repetitive construct can form functional HACs without CENP-B or initial CENP-A nucleosome seeding, revealing distinct paths to centromere formation for different DNA sequence types. Our developments streamline the construction and characterization of HACs to facilitate mammalian synthetic genome efforts.
Assuntos
Centrômero/metabolismo , Cromossomos Artificiais Humanos/metabolismo , DNA Satélite/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Centrômero/genética , Proteína Centromérica A/genética , Proteína Centromérica A/metabolismo , Proteína B de Centrômero/deficiência , Proteína B de Centrômero/genética , Proteína B de Centrômero/metabolismo , Epigênese Genética , Humanos , Nucleossomos/química , Nucleossomos/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismoRESUMO
The targeting of telomerase and telomere maintenance mechanisms represents a promising therapeutic approach for various types of cancer. In this work, we designed a new protocol to screen for and rank the efficacy of compounds specifically targeting telomeres and telomerase. This approach used two isogenic cell lines containing a circular human artificial chromosome (HAC, lacking telomeres) and a linear HAC (containing telomeres) marked with the EGFP transgene; compounds that target telomerase or telomeres should preferentially induce loss of the linear HAC but not the circular HAC. Our assay allowed quantification of chromosome loss by routine flow cytometry. We applied this dual-HAC assay to rank a set of known and newly developed compounds, including G-quadruplex (G4) ligands. Among the latter group, two compounds, Cu-ttpy and Pt-ttpy, induced a high rate of linear HAC loss with no significant effect on the mitotic stability of a circular HAC. Analysis of the mitotic phenotypes induced by these drugs revealed an elevated rate of chromatin bridges in late mitosis and cytokinesis as well as UFB (ultrafine bridges). Chromosome loss after Pt-ttpy or Cu-ttpy treatment correlated with the induction of telomere-associated DNA damage. Overall, this platform enables identification and ranking of compounds that greatly increase chromosome mis-segregation rates as a result of telomere dysfunction and may expedite the development of new therapeutic strategies for cancer treatment.Significance: An assay provides a unique opportunity to screen thousands of chemical compounds for their ability to inactivate replication of telomeric ends in cancer cells and holds potential to lay the foundation for the discovery of new treatments for cancer. Cancer Res; 78(21); 6282-96. ©2018 AACR.
Assuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Telomerase/antagonistas & inibidores , Telômero/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Cromatina , Cromossomos , Cromossomos Artificiais Humanos , Dano ao DNA , Desenho de Fármacos , Células HCT116 , Humanos , Ácidos Hidroxâmicos/farmacologia , Mitose , Neoplasias/genética , TransgenesRESUMO
Since their description in the late 1990s, Human Artificial Chromosomes (HACs) bearing functional kinetochores have been considered as promising systems for gene delivery and expression. More recently a HAC assembled from a synthetic alphoid DNA array has been exploited in studies of centromeric chromatin and in assessing the impact of different epigenetic modifications on kinetochore structure and function in human cells. This HAC was termed the alphoidtetO-HAC, as the synthetic monomers each contained a tetO sequence in place of the CENP-B box that can be targeted specifically with tetR-fusion proteins. Studies in which the kinetochore chromatin of the alphoidtetO-HAC was specifically modified, revealed that heterochromatin is incompatible with centromere function and that centromeric transcription is important for centromere assembly and maintenance. In addition, the alphoidtetO-HAC was modified to carry large gene inserts that are expressed in target cells under conditions that recapitulate the physiological regulation of endogenous loci. Importantly, the phenotypes arising from stable gene expression can be reversed when cells are "cured" of the HAC by inactivating its kinetochore in proliferating cell populations, a feature that provides a control for phenotypic changes attributed to expression of HAC-encoded genes. AlphoidtetO-HAC-based technology has also been used to develop new drug screening and assessment strategies to manipulate the CIN phenotype in cancer cells. In summary, the alphoidtetO-HAC is proving to be a versatile tool for studying human chromosome transactions and structure as well as for genome and cancer studies.
Assuntos
Centrômero/metabolismo , Cromossomos Artificiais Humanos/genética , Neoplasias/patologia , Animais , Proteína B de Centrômero/genética , Proteína B de Centrômero/metabolismo , Instabilidade Cromossômica , Cromossomos Artificiais Humanos/metabolismo , Técnicas de Transferência de Genes , Histonas/metabolismo , Humanos , Neoplasias/genéticaRESUMO
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 (alphoidtetO-HAC). We have demonstrated that the IIS-alphoidtetO-HAC system is a valuable genetic tool by reassembling a functional gene from multiple segments on the HAC. IIS-alphoidtetO-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 markers at each DNA insertion step, simplifying selection of correct clones; and presence of an error proofing mechanism to remove cells with misincorporated DNA segments, which improves the integrity of assembly. In addition, the IIS-alphoidtetO-HAC carrying a locus of interest is removable, offering the unique possibility to revert the cell line to its pretransformed state and compare the phenotypes of human cells with and without a functional copy of a gene(s). Thus, IIS-alphoidtetO-HAC allows investigation of complex biomedical pathways, gene(s) regulation, and has the potential to engineer synthetic chromosomes with a predetermined set of genes.
Assuntos
Cromossomos Artificiais Humanos/genética , DNA/metabolismo , Integrases/genética , Cinetocoros/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , DNA/genética , Humanos , Hibridização in Situ Fluorescente , Integrases/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Recombinação Genética , Proteína Supressora de Tumor Von Hippel-Lindau/genéticaRESUMO
Transferring large or multiple genes into primary human stem/progenitor cells is challenged by restrictions in vector capacity, and this hurdle limits the success of gene therapy. A paradigm is Duchenne muscular dystrophy (DMD), an incurable disorder caused by mutations in the largest human gene: dystrophin. The combination of large-capacity vectors, such as human artificial chromosomes (HACs), with stem/progenitor cells may overcome this limitation. We previously reported amelioration of the dystrophic phenotype in mice transplanted with murine muscle progenitors containing a HAC with the entire dystrophin locus (DYS-HAC). However, translation of this strategy to human muscle progenitors requires extension of their proliferative potential to withstand clonal cell expansion after HAC transfer. Here, we show that reversible cell immortalisation mediated by lentivirally delivered excisable hTERT and Bmi1 transgenes extended cell proliferation, enabling transfer of a novel DYS-HAC into DMD satellite cell-derived myoblasts and perivascular cell-derived mesoangioblasts. Genetically corrected cells maintained a stable karyotype, did not undergo tumorigenic transformation and retained their migration ability. Cells remained myogenic in vitro (spontaneously or upon MyoD induction) and engrafted murine skeletal muscle upon transplantation. Finally, we combined the aforementioned functions into a next-generation HAC capable of delivering reversible immortalisation, complete genetic correction, additional dystrophin expression, inducible differentiation and controllable cell death. This work establishes a novel platform for complex gene transfer into clinically relevant human muscle progenitors for DMD gene therapy.
Assuntos
Cromossomos Artificiais Humanos , Distrofina/genética , Terapia Genética/métodos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Animais , Células Cultivadas , Vetores Genéticos , Humanos , Camundongos , Modelos Animais , MutaçãoRESUMO
Most solid tumors are aneuploid, carrying an abnormal number of chromosomes, and they frequently missegregate whole chromosomes in a phenomenon termed chromosome instability (CIN). While CIN can be provoked through disruption of numerous mitotic pathways, it is not clear which of these mechanisms are most critical, or whether alternative mechanisms could also contribute significantly in vivo. One difficulty in determining the relative importance of candidate CIN regulators has been the lack of a straightforward, quantitative assay for CIN in live human cells: While gross mitotic abnormalities can be detected visually, moderate levels of CIN may not be obvious, and are thus problematic to measure. To address this issue, we have developed the first Human Artificial Chromosome (HAC)-based quantitative live-cell assay for mitotic chromosome segregation in human cells. We have produced U2OS-Phoenix cells carrying the alphoid(tetO)-HAC encoding copies of eGFP fused to the destruction box (DB) of anaphase promoting complex/cyclosome (APC/C) substrate hSecurin and sequences encoding the tetracycline repressor fused to mCherry (TetR-mCherry). Upon HAC missegregation, daughter cells that do not obtain a copy of the HAC are GFP negative in the subsequent interphase. The HAC can also be monitored live following the TetR-mCherry signal. U2OS-Phoenix cells show low inherent levels of CIN, which can be enhanced by agents that target mitotic progression through distinct mechanisms. This assay allows direct detection of CIN induced by clinically important agents without conspicuous mitotic defects, allowing us to score increased levels of CIN that fall below the threshold required for discernable morphological disruption.
Assuntos
Antineoplásicos/farmacologia , Instabilidade Cromossômica/genética , Mitose/genética , Animais , Células CHO , Linhagem Celular Tumoral , Separação Celular , Instabilidade Cromossômica/efeitos dos fármacos , Segregação de Cromossomos/efeitos dos fármacos , Cromossomos Artificiais Humanos/genética , Cricetinae , Cricetulus , Proteínas de Fluorescência Verde/metabolismo , Humanos , Mitose/efeitos dos fármacosRESUMO
Accumulating data indicates that chromosome instability (CIN) common to cancer cells can be used as a target for cancer therapy. At present the rate of chromosome mis-segregation is quantified by laborious techniques such as coupling clonal cell analysis with karyotyping or fluorescence in situ hybridization (FISH). Recently, a novel assay was developed based on the loss of a non-essential human artificial chromosome (HAC) carrying a constitutively expressed EGFP transgene ("loss of signal" assay). Using this system, anticancer drugs can be easily ranked on by their effect on HAC loss. However, it is problematic to covert this "loss of signal" assay into a high-throughput screen to identify drugs and mutations that increase CIN levels. To address this point, we re-designed the HAC-based assay. In this new system, the HAC carries a constitutively expressed shRNA against the EGFP transgene integrated into human genome. Thus, cells that inherit the HAC display no green fluorescence, while cells lacking the HAC do. We verified the accuracy of this "gain of signal" assay by measuring the level of CIN induced by known antimitotic drugs and added to the list of previously ranked CIN inducing compounds, two newly characterized inhibitors of the centromere-associated protein CENP-E, PF-2771 and GSK923295 that exhibit the highest effect on chromosome instability measured to date. The "gain of signal" assay was also sensitive enough to detect increase of CIN after siRNA depletion of known genes controlling mitotic progression through distinct mechanisms. Hence this assay can be utilized in future experiments to uncover novel human CIN genes, which will provide novel insight into the pathogenesis of cancer. Also described is the possible conversion of this new assay into a high-throughput screen using a fluorescence microplate reader to characterize chemical libraries and identify new conditions that modulate CIN level.
Assuntos
Bioensaio/métodos , Instabilidade Cromossômica , Cromossomos Artificiais Humanos/genética , Fibrossarcoma/diagnóstico , Fibrossarcoma/genética , Proteínas de Fluorescência Verde/metabolismo , Apoptose , Proliferação de Células , Proteínas de Fluorescência Verde/genética , Humanos , Hibridização in Situ Fluorescente , Células Tumorais CultivadasRESUMO
BACKGROUND: Human artificial chromosome (HAC) vectors have some unique characteristics as compared with conventional vectors, carrying large transgenes without size limitation, showing persistent expression of transgenes, and existing independently from host genome in cells. With these features, HACs are expected to be promising vectors for modifications of a variety of cell types. However, the method of introduction of HACs into target cells is confined to microcell-mediated chromosome transfer (MMCT), which is less efficient than other methods of vector introduction. Application of Measles Virus (MV) fusogenic proteins to MMCT instead of polyethylene glycol (PEG) has partly solved this drawback, whereas the tropism of MV fusogenic proteins is restricted to human CD46- or SLAM-positive cells. RESULTS: Here, we show that retargeting of microcell fusion by adding anti-Transferrin receptor (TfR) single chain antibodies (scFvs) to the extracellular C-terminus of the MV-H protein improves the efficiency of MV-MMCT to human fibroblasts which originally barely express both native MV receptors, and are therefore resistant to MV-MMCT. Efficacy of chimeric fusogenic proteins was evaluated by the evidence that the HAC, tagged with a drug-resistant gene and an EGFP gene, was transferred from CHO donor cells into human fibroblasts. Furthermore, it was demonstrated that no perturbation of either the HAC status or the functions of transgenes was observed on account of retargeted MV-MMCT when another HAC carrying four reprogramming factors (iHAC) was transferred into human fibroblasts. CONCLUSIONS: Retargeted MV-MMCT using chimeric H protein with scFvs succeeded in extending the cell spectrum for gene transfer via HAC vectors. Therefore, this technology could facilitate the systematic cell engineering by HACs.
Assuntos
Cromossomos Artificiais Humanos/genética , Vírus do Sarampo/genética , Proteínas Virais de Fusão/genética , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Fibroblastos , Técnicas de Transferência de Genes , Humanos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Large-scale production of fully human IgG (hIgG) or human polyclonal antibodies (hpAbs) by transgenic animals could be useful for human therapy. However, production level of hpAbs in transgenic animals is generally very low, probably due to the fact that evolutionarily unique interspecies-incompatible genomic sequences between human and non-human host species may impede high production of fully hIgG in the non-human environment. To address this issue, we performed species-specific human artificial chromosome (HAC) engineering and tested these engineered HAC in cattle. Our previous study has demonstrated that site-specific genomic chimerization of pre-B cell receptor/B cell receptor (pre-BCR/BCR) components on HAC vectors significantly improves human IgG expression in cattle where the endogenous bovine immunoglobulin genes were knocked out. In this report, hIgG1 class switch regulatory elements were subjected to site-specific genomic chimerization on HAC vectors to further enhance hIgG expression and improve hIgG subclass distribution in cattle. These species-specific modifications in a chromosome scale resulted in much higher production levels of fully hIgG of up to 15 g/L in sera or plasma, the highest ever reported for a transgenic animal system. Transchromosomic (Tc) cattle containing engineered HAC vectors generated hpAbs with high titers against human-origin antigens following immunization. This study clearly demonstrates that species-specific sequence differences in pre-BCR/BCR components and IgG1 class switch regulatory elements between human and bovine are indeed functionally distinct across the two species, and therefore, are responsible for low production of fully hIgG in our early versions of Tc cattle. The high production levels of fully hIgG with hIgG1 subclass dominancy in a large farm animal species achieved here is an important milestone towards broad therapeutic applications of hpAbs.
Assuntos
Anticorpos Monoclonais Humanizados/biossíntese , Anticorpos Monoclonais/biossíntese , Cromossomos Artificiais Humanos/imunologia , Vetores Genéticos/metabolismo , Imunoglobulina G/biossíntese , Receptores de Células Precursoras de Linfócitos B/imunologia , Animais , Animais Geneticamente Modificados , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/imunologia , Antígenos/química , Antígenos/imunologia , Bovinos , Linhagem Celular Tumoral , Galinhas , Mapeamento Cromossômico , Cromossomos Artificiais Humanos/química , Técnicas de Inativação de Genes , Engenharia Genética , Vetores Genéticos/química , Humanos , Imunização , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Linfócitos/citologia , Linfócitos/imunologia , Receptores de Células Precursoras de Linfócitos B/genética , Especificidade da EspécieRESUMO
De novo artificial chromosomes expressing genes have been generated in human embryonic stem cells (hESc) and are maintained following differentiation into other cell types. Human artificial chromosomes (HAC) are small, functional, extrachromosomal elements, which behave as normal chromosomes in human cells. De novo HAC are generated following delivery of alpha satellite DNA into target cells. HAC are characterized by high levels of mitotic stability and are used as models to study centromere formation and chromosome organisation. They are successful and effective as gene expression vectors since they remain autonomous and can accommodate larger genes and regulatory regions for long-term expression studies in cells unlike other viral gene delivery vectors currently used. Transferring the essential DNA sequences for HAC formation intact across the cell membrane has been challenging for a number of years. A highly efficient delivery system based on HSV-1 amplicons has been used to target DNA directly to the ES cell nucleus and HAC stably generated in human embryonic stem cells (hESc) at high frequency. HAC were detected using an improved protocol for hESc chromosome harvesting, which consistently produced high-quality metaphase spreads that could routinely detect HAC in hESc. In tumour cells, the input DNA often integrated in the host chromosomes, but in the host ES genome, it remained intact. The hESc containing the HAC formed embryoid bodies, generated teratoma in mice, and differentiated into neuronal cells where the HAC were maintained. The HAC structure and chromatin composition was similar to the endogenous hESc chromosomes. This review will discuss the technological advances in HAC vector delivery using HSV-1 amplicons and the improvements in the identification of de novo HAC in hESc.
Assuntos
Cromossomos Artificiais Humanos/genética , Técnicas de Transferência de Genes , Terapia Genética/métodos , Herpesvirus Humano 1/genética , Células-Tronco Embrionárias , Vetores Genéticos/genética , Humanos , Hibridização in Situ FluorescenteRESUMO
De novo assembled alphoid(tetO)-type human artificial chromosomes (HACs) represent a novel promising generation of high capacity episomal vectors. Their function and persistence, and any adverse effects, in various cell types in live animals, have not, however, been explored. In this study we transferred the alphoid(tetO)-HAC into mouse ES cells and assessed whether the presence of this extra chromosome affects their pluripotent properties. Alphoid(tetO)-HAC-bearing ES cells were indistinguishable from their wild-type counterparts: they retained self-renewal potential and full capacity for multilineage differentiation during mouse development, whereas the HAC itself was mitotically and transcriptionally stable during this process. Our data provide the first example of fully synthetic DNA behaving like a normal chromosome in cells of living animals. It also opens a new perspective into functional genetic studies in laboratory animals as well as stem cell-based regenerative medicine.
Assuntos
Diferenciação Celular , Cromossomos Artificiais Humanos/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Células CHO , Cromossomos Artificiais Humanos/genética , Cricetinae , Cricetulus , Feminino , Técnicas de Transferência de Genes , Terapia Genética , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/citologia , Teratoma/metabolismo , Teratoma/patologiaRESUMO
Human artificial chromosome (HAC)-based vectors represent an alternative technology for gene delivery and expression with a potential to overcome the problems caused by virus-based vectors. The recently developed alphoid(tetO)-HAC has an advantage over other HAC vectors because it can be easily eliminated from cells by inactivation of the HAC kinetochore via binding of chromatin modifiers, tTA or tTS, to its centromeric tetO sequences. This provides a unique control for phenotypes induced by genes loaded into the HAC. The alphoid(tetO)-HAC elimination is highly efficient when a high level of chromatin modifiers as tetR fusion proteins is achieved following transfection of cells by a retrovirus vector. However, such vectors are potentially mutagenic and might want to be avoided under some circumstances. Here, we describe a novel system that allows verification of phenotypic changes attributed to expression of genes from the HAC without a transfection step. We demonstrated that a single copy of tTA(VP64) carrying four tandem repeats of the VP16 domain constitutively expressed from the HAC is capable to generate chromatin changes in the HAC kinetochore that are not compatible with its function. To adopt the alphoid(tetO)-HAC for routine gene function studies, we constructed a new TAR-BRV- tTA(VP64) cloning vector that allows a selective isolation of a gene of interest from genomic DNA in yeast followed by its direct transfer to bacterial cells and subsequent loading into the loxP site of the alphoid(tetO)-HAC in hamster CHO cells from where the HAC may be MMCT-transferred to the recipient human cells.