RESUMEN
The luciferase reporter assay has become one of the conventional methods for cytotoxicity evaluation. Typically, the decrease of luminescence expressed by a constitutive promoter is used as an index of cytotoxicity. However, to our knowledge, there have been no reports of the correlation between cytotoxicity and luminescence intensity. In this study, to accurately verify the correlation between them, beetle luciferase was stably expressed in human hepatoma HepG2 cells harboring the multi-integrase mouse artificial chromosome vector. We showed that the cytotoxicity assay using luciferase does not depend on the stability of luciferase protein and the kind of constitutive promoter. Next, HepG2 cells in which green-emitting beetle luciferase was expressed under the control of CAG promoter were exposed to 58 compounds. The luminescence intensity and cytotoxicity curves of cells exposed to 48 compounds showed similar tendencies, whereas those of cells exposed to 10 compounds did not do so, although the curves gradually approached each other with increasing exposure time. Finally, we demonstrated that luciferase expressed under the control of a constitutive promoter can be utilized both as an internal control reporter for normalizing a test reporter and for monitoring cytotoxicity when two kinds of luciferases are simultaneously used in the cytotoxicity assay.
Asunto(s)
Cromosomas Artificiales de los Mamíferos , Proteínas de Insectos , Luciferasas , Mediciones Luminiscentes/métodos , Regiones Promotoras Genéticas , Animales , Cromosomas Artificiales de los Mamíferos/genética , Cromosomas Artificiales de los Mamíferos/metabolismo , Escarabajos , Células Hep G2 , Humanos , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Pruebas de Toxicidad/métodosRESUMEN
The current applications for cat cloning include production of models for the study of human and animal diseases. This study was conducted to investigate the optimal fusion protocol on in vitro development of transgenic cloned cat embryos by comparing duration of electric pulse. Cat fibroblast cells containing a human artificial chromosome (HAC) vector were used as genetically modified nuclear donor cells. Couplets were fused and activated simultaneously with a single DC pulse of 3.0 kV/cm for either 30 or 60 µs. Low rates of fusion and embryo development to the blastocyst stage were observed in the reconstructed HAC-transchromosomic embryos, when the duration of fusion was prolonged to 60 µs. In contrast, the prolongation of electric pulse duration improved the embryo development and quality in the reconstructed control embryos without HAC vector. Our results suggested that the optimal parameters of electric pulses for fusion in cat somatic cell nuclear transfer vary among the types used for donor cells.
Asunto(s)
Gatos/embriología , Cromosomas Artificiales Humanos , Clonación de Organismos/veterinaria , Animales , Gatos/genética , Desarrollo Embrionario , Femenino , Masculino , Técnicas de Transferencia Nuclear/veterinariaRESUMEN
Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance, and the ability to carry large gene inserts. We previously developed HAC vectors from the normal human chromosomes using a chromosome engineering technique. However, endogenous genes were remained in these HACs, limiting their therapeutic applications. In this study, we refined a HAC vector without endogenous genes from human chromosome 21 in homologous recombination-proficient chicken DT40 cells. The HAC was physically characterized using a transformation-associated recombination (TAR) cloning strategy followed by sequencing of TAR-bacterial artificial chromosome clones. No endogenous genes were remained in the HAC. We demonstrated that any desired gene can be cloned into the HAC using the Cre-loxP system in Chinese hamster ovary cells, or a homologous recombination system in DT40 cells. The HAC can be efficiently transferred to other type of cells including mouse ES cells via microcell-mediated chromosome transfer. The transferred HAC was stably maintained in vitro and in vivo. Furthermore, tumor cells containing a HAC carrying the suicide gene, herpes simplex virus thymidine kinase (HSV-TK), were selectively killed by ganciclovir in vitro and in vivo. Thus, this novel HAC vector may be useful not only for gene and cell therapy, but also for animal transgenesis.
Asunto(s)
Cromosomas Artificiales Humanos , Terapia Genética/métodos , Vectores Genéticos , Animales , Línea Celular , Cromosomas Humanos Par 21 , Clonación Molecular , Técnicas de Transferencia de Gen , Humanos , Ratones , Recombinación GenéticaRESUMEN
Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance that avoids insertional mutations and the ability to carry large gene inserts including regulatory elements. Multipotent germline stem (mGS) cells have a great potential for gene therapy because they can be generated from an individual's testes, and when reintroduced can contribute to the specialized function of any tissue. As a proof of concept, we herein report the functional restoration of a genetic deficiency in mouse p53-/- mGS cells, using a HAC with a genomic human p53 gene introduced via microcell-mediated chromosome transfer. The p53 phenotypes of gene regulation and radiation sensitivity were complemented by introducing the p53-HAC and the cells differentiated into several different tissue types in vivo and in vitro. Therefore, the combination of using mGS cells with HACs provides a new tool for gene and cell therapies. The next step is to demonstrate functional restoration using animal models for future gene therapy.
Asunto(s)
Cromosomas Artificiales Humanos , Genes p53 , Terapia Genética/métodos , Células Madre Multipotentes/metabolismo , Teratoma/terapia , Animales , Células CHO , Diferenciación Celular , Células Cultivadas , Clonación Molecular , Cricetinae , Cricetulus , Células Madre Embrionarias/metabolismo , Femenino , Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Masculino , Ratones , Ratones Transgénicos , Células Madre Multipotentes/citología , Trasplante de Neoplasias , Transfección/métodos , TransgenesRESUMEN
Trisomy 21 (Ts21) is the most common live-born human aneuploidy and results in a constellation of features known as Down syndrome (DS). Ts21 is a frequent cause of congenital heart defects and the leading genetic cause of mental retardation. Although overexpression of a gene(s) or gene cluster on human chromosome 21 (Chr 21) or the genome imbalance by Ts21 has been suggested to play a key role in bringing about the diverse DS phenotypes, little is known about the molecular mechanisms underlying the various phenotypes associated with DS. Four approaches have been used to model DS to investigate the gene dosage effects of an extra copy of Chr 21 on various phenotypes; 1) Transgenic mice overexpressing a single gene from Chr 21, 2) YAC/BAC/PAC transgenic mice containing a single gene or genes on Chr 21, 3) Mice with intact/partial trisomy 16, a region with homology to human Chr 21 and 4) Human Chr 21 transchromosomal (Tc) mice. Here we review our new model system for the study of DS using the Tc technology, including the biological effects of an additional Chr 21 in vivo and in vitro.
Asunto(s)
Cromosomas Humanos Par 21/genética , Modelos Animales de Enfermedad , Síndrome de Down/genética , Animales , Quimera/genética , Humanos , Ratones , Ratones Transgénicos , FenotipoRESUMEN
This article summarizes our efforts to use chromosome-based vectors for animal transgenesis, which may have a benefit for overcoming the size constraints of cloned transgenes in conventional techniques. Since the initial trial for introducing naturally occurring human chromosome fragments (hCFs) with large and complex immunogulobulin (Ig) loci into mice we have obtained several lines of trans-chromosomic (Tc) mice with transmittable hCFs. As expected the normal tissue-specific expression of introduced human genes was reproduced in them by inclusion of essential remote regulatory elements. Recent development of 'chromosome cloning' technique that enable construction of human artificial chromosomes (HACs) containing a defined chromosomal region should prevent the introduction of additional genes other than genes of interest and thus enhance the utility of chromosome vector system. Using this technique a panel of HACs harboring inserts ranging in size from 1.5 to 10 Mb from three human chromosomes (hChr2, 7, 22) has been constructed. Tc animals containing the HACs may be valuable not only as a powerful tool for functional genomics but also as an in vivo model to study therapeutic gene delivery by HACs.
Asunto(s)
Cromosomas Artificiales , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Animales , Ratones , Ratones TransgénicosRESUMEN
We generated transchromosomal (Tc) mice containing a human chromosome 21 fragment (hCF21) using mouse embryonic stem (ES) cells with the transferred hCF21. Here we report breeding analyses that test the maintenance rate of the hCF21 in Tc mice of two different genetic backgrounds, MCH (ICR) and C57BL/6. Fluorescence in situ hybridization and polymerase chain reaction-based DNA analyses revealed that the structure of the hCF21 fragment including the CBR1, SIM2, HLCS, and D21S268 markers, was approximately 5 Mb in size, and was transmitted at least to the F3 generation. Though the retention rate of the hCF21 was variable among individual mice, for example, 21%-92% in brain and 10%-92% in tail fibroblasts, the C57BL/6 background yielded a higher retention rate than did the MCH (ICR). These results suggest that the hCF21 could be maintained stably in Tc mice, depending on the genetic background. The panel of Tc mice will be a useful model to investigate the function of genes on the hCF21 fragment in various tissues through germinal transmission.
Asunto(s)
Cromosomas Humanos Par 21 , Ratones Transgénicos , Animales , Técnicas de Transferencia de Gen , Marcadores Genéticos , Humanos , Hibridación Fluorescente in Situ , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Modelos Genéticos , Reacción en Cadena de la PolimerasaRESUMEN
Trisomy 21 (Ts21) is the most common live-born human aneuploidy; it results in a constellation of features known as Down's syndrome (DS). Ts21 is the most frequent cause of congenital heart defects and the leading genetic cause of mental retardation. To investigate the gene dosage effects of an extra copy of human chromosome 21 (Chr 21) on various phenotypes, we used microcell-mediated chromosome transfer to create embryonic stem (ES) cells containing Chr 21. ES cell lines retaining Chr 21 as an independent chromosome were used to produce chimeric mice with a substantial contribution from Chr 21-containing cells. Fluorescence in situ hybridization and PCR-based DNA analysis revealed that Chr 21 was substationally intact but had sustained a small deletion. The freely segregating Chr 21 was lost during development in some tissues, resulting in a panel of chimeric mice with various mosaicism as regards retention of the Chr 21. These chimeric mice showed a high correlation between retention of Chr 21 in the brain and impairment in learning or emotional behavior by open-field, contextual fear conditioning and forced swim tests. Hypoplastic thymus and cardiac defects, i.e. double outlet right ventricle and riding aorta, were observed in a considerable number of chimeric mouse fetuses with a high contribution of Chr 21. These chimeric mice mimic a wide variety of phenotypic traits of DS, revealing the utility of mice containing Chr 21 as unique models for DS and for the identification of genes responsible for DS.
Asunto(s)
Conducta Animal , Cromosomas Humanos Par 21/genética , Síndrome de Down/genética , Cardiopatías Congénitas/genética , Trastornos Mentales/genética , Animales , Quimera/genética , ADN/análisis , Cartilla de ADN/química , Síndrome de Down/patología , Femenino , Cardiopatías Congénitas/patología , Humanos , Hibridación Fluorescente in Situ , Trastornos Mentales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Actividad Motora , Mutación , Miocardio/metabolismo , Miocardio/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/metabolismo , Células Madre/patologíaRESUMEN
For introducing regions of human chromosomes greater than a megabase into cells or animals, we have developed a chromosome-cloning system in which defined regions of human chromosomes can be cloned into a stable human minichromosome vector in homologous recombination-proficient chicken DT40 cells. The stable minichromosome vector allowed a 10 Mb-sized region of the mitotically unstable human chromosome 22 to be stably maintained in mouse embryonic stem (ES) cells, and in mice. Furthermore, we demonstrated functional expression of human genes from the HAC in mice. This study describes a stable cloning and expression system for greater than megabase-sized regions of human chromosomes.