RESUMEN
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. Now they are being modified and developed for gene therapy both ex vivo and in vivo. The advantages of using HAC vectors are that they remain autonomous and behave as a normal chromosome. They are attractive for therapy studies without the harmful consequences of integration of exogenous DNA into host chromosomes. HAC vectors are also the only autonomous stable vectors that accommodate large sequences (>100 kb) compared to other vectors. The challenges of manipulating these vectors for efficient delivery of genes into human cells is still ongoing, but we have made advances in transfer of gene expressing HAC vectors using the helper free (HF) amplicon vector technology for generating de novo HAC in human cells. Efficient multigene delivery was successfully achieved following simultaneous infection with two HF amplicons in a single treatment and the input DNA recombined to form a de novo HAC. Potentially several amplicons containing gene expressing HAC vectors could be transduced simultaneously which would increase the gene loading capacity of the vectors for delivery and studying full expression in human cells.
Asunto(s)
Cromosomas Artificiales/genética , Terapia Genética/métodos , Técnicas de Transferencia de Gen , HumanosRESUMEN
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 (HAC) vectors into the nucleus of human cells. The HSV-1 amplicon lacks the signals for cleavage and replication of its own genome, yet each amplicon has the capacity to incorporate up to 150 kb of exogenous DNA. In this study, we investigated whether the capacity of gene delivery could be increased by simultaneously introducing multiple HSV-1 modified amplicons carrying a gene expressing HAC vector into cells with the aim of generating a single artificial chromosome containing the desired genes. Following co-transduction of two HSV-1 HAC amplicons, artificial chromosomes were successfully generated containing the introduced genes, which were appropriately expressed in different human cell types.
Asunto(s)
Cromosomas Artificiales Humanos/genética , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Herpesvirus Humano 1/genética , Terapia Genética , HumanosRESUMEN
We analyzed the effect of transcribed noncoding RNA centromeric satellites on chromosome segregation in normal human and murine stem and fibrosarcoma cells. The overexpression of different centromeric alphoid DNAs in all cell lines induced a marked increase in chromosome mis-segregation in anaphase. Overexpression of centromeric mouse minor satellite also increased chromosome instability in the murine stem but not in human cells. Analysis of chromosome segregation in vivo showed disturbances in the mitotic progression, which was frequently unresolved. Live cell imaging revealed that overexpression of centromeric satellites resulted in several different chromosomal morphological errors in the cell nuclei. Our findings correlated with other reports that several centromeric noncoding RNAs are detected in different carcinoma cells and their expression resulted in segregation errors. Our study furnishes further insights into a novel source of genomic instability in human and murine cells. It has recently been shown that noncoding centromeric RNAs are present in some form of cancer, and thus, overexpression of several types of centromeric noncoding RNAs may be useful as a specific maker for neoplastic cells.
Asunto(s)
Inestabilidad Cromosómica , ARN no Traducido/genética , Células Madre/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Centrómero/metabolismo , Segregación Cromosómica , Humanos , Ratones , Células Madre/citologíaRESUMEN
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.
Asunto(s)
Cromosomas Artificiales Humanos/genética , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Herpesvirus Humano 1/genética , Células Madre Embrionarias , Vectores Genéticos/genética , Humanos , Hibridación Fluorescente in SituRESUMEN
We present a novel and efficient non-integrating gene expression system in human embryonic stem cells (hESc) utilizing human artificial chromosomes (HAC), which behave as autonomous endogenous host chromosomes and segregate correctly during cell division. HAC are important vectors for investigating the organization and structure of the kinetochore, and gene complementation. HAC have so far been obtained in immortalized or tumour-derived cell lines, but never in stem cells, thus limiting their potential therapeutic application. In this work, we modified the herpes simplex virus type 1 amplicon system for efficient transfer of HAC DNA into two hESc. The deriving stable clones generated green fluorescent protein gene-expressing HAC at high frequency, which were stably maintained without selection for 3 months. Importantly, no integration of the HAC DNA was observed in the hESc lines, compared with the fibrosarcoma-derived control cells, where the exogenous DNA frequently integrated in the host genome. The hESc retained pluripotency, differentiation and teratoma formation capabilities. This is the first report of successfully generating gene expressing de novo HAC in hESc, and is a significant step towards the genetic manipulation of stem cells and potential therapeutic applications.
Asunto(s)
Cromosomas Artificiales Humanos/metabolismo , Células Madre Embrionarias/metabolismo , Neuronas/citología , Neuronas/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Línea Celular , Cromosomas Artificiales Humanos/genética , Células Madre Embrionarias/citología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Herpesvirus Humano 1/genética , HumanosRESUMEN
Prolonged in vitro culture of human embryonic stem (hES) cells can result in chromosomal abnormalities believed to confer a selective advantage. This potential occurrence has crucial implications for the appropriate use of hES cells for research and therapeutic purposes. In view of this, time-point karyotypic evaluation to assess genetic stability is recommended as a necessary control test to be carried out during extensive 'passaging'. Standard techniques currently used for the cytogenetic assessment of ES cells include G-banding and/or Fluorescence in situ Hybridization (FISH)-based protocols for karyotype analysis, including M-FISH and SKY. Critical for both banding and FISH techniques are the number and quality of metaphase spreads available for analysis at the microscope. Protocols for chromosome preparation from hES and human induced pluripotent stem (hiPS) cells published so far appear to differ considerably from one laboratory to another. Here we present an optimized technique, in which both the number and the quality of chromosome metaphase spreads were substantially improved when compared to current standard techniques for chromosome preparations. We believe our protocol represents a significant advancement in this line of work, and has the required attributes of simplicity and consistency to be widely accepted as a reference method for high quality, fast chromosomal analysis of human ES and iPS cells.
Asunto(s)
Células Madre Embrionarias/clasificación , Células Madre Pluripotentes Inducidas/clasificación , Cariotipificación/métodos , Técnicas de Cultivo de Célula , Cromosomas Humanos , Demecolcina/química , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Humanos , Hibridación Fluorescente in Situ , Indicadores y Reactivos/química , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Nocodazol/química , Coloración y Etiquetado/métodosRESUMEN
The replication timing of different DNA sequences in the mammalian cell nucleus is a tightly regulated system, which affects important cellular processes such as genes expression, chromatin epigenetic marking, and maintenance of chromosome structure. For this reason, it is important to study the replication properties of specific sequences, to determine for example, if the replication timing varies in different tissues, or in the presence of specific reagents, such as hormones, or other biologically active molecules. In this chapter, we present a technique, which allows identification of specific DNA sequences by fluorescence in situ hybridization (FISH) and simultaneously analyses the incorporation of a thymidine analogue, 5-bromo-2-deoxyuridine (BrdU), to mark DNA replication. First, tissue culture cells are synchronized at the beginning of the S-phase. BrdU is then added, either at specific time-points during S-phase or during the whole of the cell cycle. After harvesting the cells, the chromosomal DNA is hybridized to FISH probes that identify specific DNA sequences; this is performed without the teratogen formamide normally used in FISH. Finally, the cell preparations are analysed with an epifluorescence microscope to determine if the sequence of interest incorporates BrdU and in which point of the S-phase.
Asunto(s)
Bromodesoxiuridina/metabolismo , ADN/genética , Hibridación Fluorescente in Situ/métodos , Afidicolina/metabolismo , Línea Celular , ADN/metabolismo , Sondas de ADN/metabolismo , Desnaturalización de Ácido Nucleico , Factores de TiempoRESUMEN
BACKGROUND: Human artificial chromosomes (HAC) are small functional extrachromosomal elements, which segregate correctly during each cell division. In human cells, they are mitotically stable, however when the HAC are transferred to murine cells they show an increased and variable rate of loss. In some cell lines the HAC are lost over a short period of time, while in others the HAC become stable without acquiring murine DNA. RESULTS: In this study, we linked the loss rate to the position of the HAC in the murine cell nucleus with respect to the chromocenters. HAC that associated preferentially with the chromocenter displayed a lower loss rate compared to the HAC that are less frequently associated. The chromocenter acts as a hub for the deposition of heterochromatic markers, controlling centromeric and pericentromeric DNA replication timing and chromosome segregation. The HAC which localized more frequently outside the chromocenters bound variable amounts of histone H3 tri-methylated at lysine 9, and the high level of intraclonal variability was associated with an increase in HAC segregation errors and delayed DNA replication timing. CONCLUSION: This is a novel result indicating that HAC segregation is closely linked to the position in the murine nucleus and gives important insight for HAC gene expression studies in murine cells and establishing murine models of human genetic disease.
Asunto(s)
Cromatina/química , Inestabilidad Cromosómica , Cromosomas Artificiales Humanos/genética , Cromosomas Artificiales Humanos/metabolismo , Animales , Línea Celular , Núcleo Celular/genética , Centrómero/fisiología , Cromatina/metabolismo , Técnicas de Transferencia de Gen , Histonas/metabolismo , Humanos , Hibridación Fluorescente in Situ , Metafase , Ratones , MitosisRESUMEN
FISH (Fluorescence in situ hybridization) is a powerful technique that detects and localises specific DNA sequences on metaphase chromosomes, interphase nuclei or chromatin fibres. When coupled to BrdU (5-Bromo 2-deoxy-uridine) labeling of newly replicated DNA, the replication properties of different DNA sequences can be analysed. However, the technique for the detection of BrdU incorporation is time consuming, and relies on acidic pH buffer treatments, that prevent use of pH sensitive fluorochromes such as FITC (Fluoro-isothiocianate) during FISH. In this work, we describe a simplified protocol that allows the simultaneous detection of FISH signals and BrdU incorporation. Since the technique does not involve paraformaldehyde for cell fixation, or formamide for denaturation of the target DNA and in post-hybridisation washes, it represents a safer alternative to classical FISH techniques.