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1.
BMC Biotechnol ; 20(1): 44, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32819341

RESUMO

BACKGROUND: Oral administration is the most common way to deliver drugs to the systemic circulation or target organs. Orally administered drugs are absorbed in the intestine and metabolized in the intestine and liver. In the early stages of drug development, it is important to predict first-pass metabolism accurately to select candidate drugs with high bioavailability. The Caco-2 cell line derived from colorectal cancer is widely used as an intestinal model to assess drug membrane permeability. However, because the expression of major drug-metabolizing enzymes, such as cytochrome P450 (CYP), is extremely low in Caco-2 cells, it is difficult to predict intestinal metabolism, which is a significant factor in predicting oral drug bioavailability. Previously, we constructed a mouse artificial chromosome vector carrying the CYP (CYP2C9, CYP2C19, CYP2D6, and CYP3A4) and P450 oxidoreductase (POR) (4CYPs-MAC) genes and increased CYP expression and metabolic activity in HepG2 cells via transfer of this vector. RESULTS: In the current study, to improve the Caco-2 cell assay model by taking metabolism into account, we attempted to increase CYP expression by transferring the 4CYPs-MAC into Caco-2 cells. The Caco-2 cells carrying the 4CYPs-MAC showed higher CYP mRNA expression and activity. In addition, high metabolic activity, availability for permeation test, and the potential to assess drug-drug interactions were confirmed. CONCLUSIONS: The established Caco-2 cells with the 4CYPs-MAC are expected to enable more accurate prediction of the absorption and metabolism in the human intestine than parental Caco-2 cells. The mammalian artificial chromosome vector system would provide useful models for drug development.


Assuntos
Cromossomos Artificiais de Mamíferos/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Administração Oral , Animais , Disponibilidade Biológica , Células CACO-2 , Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP2C9/genética , Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Expressão Gênica , Células Hep G2 , Humanos , Fígado/metabolismo , Camundongos , RNA Mensageiro/metabolismo
2.
Exp Cell Res ; 390(2): 111914, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32142854

RESUMO

"Genomically" humanized animals are invaluable tools for generating human disease models and for biomedical research. Humanized animal models have generally been developed via conventional transgenic technologies; however, conventional gene delivery vectors such as viruses, plasmids, bacterial artificial chromosomes, P1 phase-derived artificial chromosomes, and yeast artificial chromosomes have limitations for transgenic animal creation as their loading gene capacity is restricted, and the expression of transgenes is unstable. Transchromosomic (Tc) techniques using mammalian artificial chromosomes, including human chromosome fragments, human artificial chromosomes, and mouse artificial chromosomes, have overcome these limitations. These tools can carry multiple genes or Mb-sized genomic loci and their associated regulatory elements, which has facilitated the creation of more useful and complex transgenic models for human disease, drug development, and humanized animal research. This review describes the history of Tc animal development, the applications of Tc animals, and future prospects.


Assuntos
Animais Geneticamente Modificados/genética , Cromossomos Artificiais de Mamíferos/química , Cromossomos Humanos/química , Edição de Genes/métodos , Técnicas de Transferência de Genes , Aneuploidia , Animais , Bovinos , Cromossomos Artificiais de Mamíferos/metabolismo , Cromossomos Humanos/metabolismo , Cabras , Humanos , Camundongos , Plasmídeos/química , Plasmídeos/metabolismo , Ratos
3.
Exp Cell Res ; 390(1): 111915, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32092294

RESUMO

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.


Assuntos
Cromossomos Artificiais de Mamíferos/genética , Técnicas de Transferência de Genes , Engenharia Genética/métodos , Animais , Mapeamento Cromossômico/métodos , Humanos
4.
Exp Cell Res ; 388(2): 111851, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-31952951

RESUMO

In the near twenty-year existence of the human and mammalian artificial chromosome field, the technologies for artificial chromosome construction and installation into desired cell types or organisms have evolved with the rest of modern molecular and synthetic biology. Medical, industrial, pharmaceutical, agricultural, and basic research scientists seek the as yet unrealized promise of human and mammalian artificial chromosomes. Existing technologies for both top-down and bottom-up approaches to construct these artificial chromosomes for use in higher eukaryotes are very different but aspire to achieve similar results. New capacity for production of chromosome sized synthetic DNA will likely shift the field towards more bottom-up approaches, but not completely. Similarly, new approaches to install human and mammalian artificial chromosomes in target cells will compete with the microcell mediated cell transfer methods that currently dominate the field.


Assuntos
Cromossomos Artificiais de Mamíferos , Cromossomos de Mamíferos/genética , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos , Animais , Humanos
5.
PLoS One ; 12(10): e0187072, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29065189

RESUMO

The utility of HepG2 cells to assess drug metabolism and toxicity induced by chemical compounds is hampered by their low cytochrome P450 (CYP) activities. To overcome this limitation, we established HepG2 cell lines expressing major CYP enzymes involved in drug metabolism (CYP2C9, CYP2C19, CYP2D6, and CYP3A4) and CYP oxidoreductase (POR) using the mammalian-derived artificial chromosome vector. Transchromosomic HepG2 (TC-HepG2) cells expressing four CYPs and POR were used to determine time- and concentration-dependent inhibition and toxicity of several compounds by luminescence detection of CYP-specific substrates and cell viability assays. Gene expression levels of all four CYPs and POR, as well as the CYP activities, were higher in TC-HepG2 clones than in parental HepG2 cells. Additionally, the activity levels of all CYPs were reduced in a concentration-dependent manner by specific CYP inhibitors. Furthermore, preincubation of TC-HepG2 cells with CYP inhibitors known as time-dependent inhibitors (TDI) prior to the addition of CYP-specific substrates determined that CYP inhibition was enhanced in the TDI group than in the non-TDI group. Finally, the IC50 of bioactivable compound aflatoxin B1 was lower in TC-HepG2 cells than in HepG2 cells. In conclusion, the TC-HepG2 cells characterized in the current study are a highly versatile model to evaluate drug-drug interactions and hepatotoxicity in initial screening of candidate drug compounds, which require a high degree of processing capacity and reliability.


Assuntos
Cromossomos Artificiais de Mamíferos , Sistema Enzimático do Citocromo P-450/genética , Inibidores Enzimáticos/farmacologia , Modelos Biológicos , Animais , Linhagem Celular , Inibidores Enzimáticos/farmacocinética , Humanos , Concentração Inibidora 50
6.
Anal Biochem ; 522: 18-29, 2017 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-28111305

RESUMO

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.


Assuntos
Cromossomos Artificiais de Mamíferos , Proteínas de Insetos , Luciferases , Medições Luminescentes/métodos , Regiões Promotoras Genéticas , Animais , Cromossomos Artificiais de Mamíferos/genética , Cromossomos Artificiais de Mamíferos/metabolismo , Besouros , Células Hep G2 , Humanos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Luciferases/genética , Luciferases/metabolismo , Camundongos , Testes de Toxicidade/métodos
7.
ACS Synth Biol ; 5(10): 1040-1049, 2016 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-27076218

RESUMO

The enabling technologies of synthetic biology are opening up new opportunities for engineering and enhancement of mammalian cells. This will stimulate diverse applications in many life science sectors such as regenerative medicine, development of biosensing cell lines, therapeutic protein production, and generation of new synthetic genetic regulatory circuits. Harnessing the full potential of these new engineering-based approaches requires the design and assembly of large DNA constructs-potentially up to chromosome scale-and the effective delivery of these large DNA payloads to the host cell. Random integration of large transgenes, encoding therapeutic proteins or genetic circuits into host chromosomes, has several drawbacks such as risks of insertional mutagenesis, lack of control over transgene copy-number and position-specific effects; these can compromise the intended functioning of genetic circuits. The development of a system orthogonal to the endogenous genome is therefore beneficial. Mammalian artificial chromosomes (MACs) are functional, add-on chromosomal elements, which behave as normal chromosomes-being replicating and portioned to daughter cells at each cell division. They are deployed as useful gene expression vectors as they remain independent from the host genome. MACs are maintained as a single-copy and can accommodate multiple gene expression cassettes of, in theory, unlimited DNA size (MACs up to 10 megabases have been constructed). MACs therefore enabled control over ectopic gene expression and represent an excellent platform to rapidly prototype and characterize novel synthetic gene circuits without recourse to engineering the host genome. This review describes the obstacles synthetic biologists face when working with mammalian systems and how the development of improved MACs can overcome these-particularly given the spectacular advances in DNA synthesis and assembly that are fuelling this research area.


Assuntos
Cromossomos Artificiais de Mamíferos/genética , Genes Sintéticos , Mamíferos/genética , Biologia Sintética , Animais , Linhagem Celular , Expressão Gênica , Engenharia Genética , Humanos , Organismos Geneticamente Modificados , Transgenes
8.
Transgenic Res ; 24(4): 717-27, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26055730

RESUMO

The mouse artificial chromosome (MAC) has several advantages as a gene delivery vector, including stable episomal maintenance of the exogenous genetic material and the ability to carry large and/or multiple gene inserts including their regulatory elements. Previously, a MAC containing multi-integration site (MI-MAC) was generated to facilitate transfer of multiple genes into desired cells. To generate transchromosomic (Tc) mice containing a MI-MAC with genes of interest, the desired genes were inserted into MI-MAC in CHO cells, and then the MI-MAC was transferred to mouse embryonic stem (mES) cells via microcell-mediated chromosome transfer (MMCT). However, the efficiency of MMCT from CHO to mES cells is very low (<10(-6)). In this study, we constructed mES cell lines containing a MI-MAC vector to directly insert a gene of interest into the MI-MAC in mES cells via a simple transfection method for Tc mouse generation. The recombination rate of the GFP gene at each attachment site (FRT, PhiC31attP, R4attP, TP901-1attP and Bxb1attP) on MI-MAC was greater than 50% in MI-MAC mES cells. Chimeric mice with high coat colour chimerism were generated from the MI-MAC mES cell lines and germline transmission from the chimera was observed. As an example for the generation of Tc mice with a desired gene by the MI-MAC mES approach, a Tc mouse strain ubiquitously expressing Emerald luciferase was efficiently established. Thus, the findings suggest that this new Tc strategy employing mES cells and a MI-MAC vector is efficient and useful for animal transgenesis.


Assuntos
Cromossomos Artificiais de Mamíferos/genética , Engenharia Genética/métodos , Vetores Genéticos/genética , Integrases/genética , Células-Tronco Embrionárias Murinas/metabolismo , Animais , Células CHO , Quimera , Cricetinae , Cricetulus , Citometria de Fluxo , Técnicas de Transferência de Genes , Células Germinativas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/citologia , Recombinação Genética , Transgenes/genética
9.
Chromosome Res ; 23(1): 143-57, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25596828

RESUMO

Mammalian artificial chromosomes (MACs) are non-integrating, autonomously replicating natural chromosome-based vectors that may carry a vast amount of genetic material, which in turn enable potentially prolonged, safe, and regulated therapeutic transgene expression and render MACs as attractive genetic vectors for "gene replacement" or for controlling differentiation pathways in target cells. Satellite-DNA-based artificial chromosomes (SATACs) can be made by induced de novo chromosome formation in cells of different mammalian and plant species. These artificially generated accessory chromosomes are composed of predictable DNA sequences, and they contain defined genetic information. SATACs have already passed a number of obstacles crucial to their further development as gene therapy vectors, including large-scale purification, transfer of purified artificial chromosomes into different cells and embryos, generation of transgenic animals and germline transmission with purified SATACs, and the tissue-specific expression of a therapeutic gene from an artificial chromosome in the milk of transgenic animals. SATACs could be used in cell therapy protocols. For these methods, the most versatile target cell would be one that was pluripotent and self-renewing to address multiple disease target cell types, thus making multilineage stem cells, such as adult derived early progenitor cells and embryonic stem cells, as attractive universal host cells.


Assuntos
Cromossomos Artificiais de Mamíferos/genética , DNA Satélite/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Mamíferos/genética , Modelos Genéticos , Células-Tronco/metabolismo , Animais , Animais Geneticamente Modificados , Humanos
10.
Acta Biol Hung ; 65(3): 331-45, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25194736

RESUMO

Direct reprogramming of mouse fibroblasts into induced pluripotent stem cells (iPS) was achieved recently by overexpression of four transcription factors encoded by retroviral vectors. Most of the virus vectors, however, may cause insertional mutagenesis in the host genome and may also induce tumor formation. Therefore, it is very important to discover novel and safer, non-viral reprogramming methods. Here we describe the reprogramming of somatic cells into iPS cells by a novel protein-based technique. Engineered Oct4, Sox2 and Klf4 transcription factors carrying an N-terminal Flag-tag and a C-terminal polyarginine tail were synthesized by a recently described mammalian artificial chromosome expression system (ACEs). This system is suitable for the high-level production of recombinant proteins in mammalian tissue culture cells. Recombinant proteins produced in this system contain all the post-translational modifications essential for the stability and the authentic function of the proteins. The engineered Oct4, Sox2 and Klf4 proteins efficiently induced the reprogramming of mouse embryonic fibroblasts by means of protein transduction. This novel method allows for the generation of iPS cells, which may be suitable for therapeutic applications in the future.


Assuntos
Reprogramação Celular , Cromossomos Artificiais de Mamíferos , Fibroblastos/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição/metabolismo , Transfecção/métodos , Animais , Células CHO , Técnicas de Cocultura , Cricetinae , Cricetulus , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/genética
11.
PLoS One ; 9(1): e85565, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24454889

RESUMO

Mammalian artificial chromosomes are natural chromosome-based vectors that may carry a vast amount of genetic material in terms of both size and number. They are reasonably stable and segregate well in both mitosis and meiosis. A platform artificial chromosome expression system (ACEs) was earlier described with multiple loading sites for a modified lambda-integrase enzyme. It has been shown that this ACEs is suitable for high-level industrial protein production and the treatment of a mouse model for a devastating human disorder, Krabbe's disease. ACEs-treated mutant mice carrying a therapeutic gene lived more than four times longer than untreated counterparts. This novel gene therapy method is called combined mammalian artificial chromosome-stem cell therapy. At present, this method suffers from the limitation that a new selection marker gene should be present for each therapeutic gene loaded onto the ACEs. Complex diseases require the cooperative action of several genes for treatment, but only a limited number of selection marker genes are available and there is also a risk of serious side-effects caused by the unwanted expression of these marker genes in mammalian cells, organs and organisms. We describe here a novel method to load multiple genes onto the ACEs by using only two selectable marker genes. These markers may be removed from the ACEs before therapeutic application. This novel technology could revolutionize gene therapeutic applications targeting the treatment of complex disorders and cancers. It could also speed up cell therapy by allowing researchers to engineer a chromosome with a predetermined set of genetic factors to differentiate adult stem cells, embryonic stem cells and induced pluripotent stem (iPS) cells into cell types of therapeutic value. It is also a suitable tool for the investigation of complex biochemical pathways in basic science by producing an ACEs with several genes from a signal transduction pathway of interest.


Assuntos
Cromossomos Artificiais de Mamíferos/genética , Genes , Vetores Genéticos , Animais , Sequência de Bases , Células CHO , Cricetinae , Cricetulus , Primers do DNA , Modelos Animais de Doenças , Hibridização in Situ Fluorescente , Camundongos , Reação em Cadeia da Polimerase
12.
Biochem Biophys Res Commun ; 442(1-2): 44-50, 2013 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-24216103

RESUMO

Human artificial chromosomes (HACs) and mouse artificial chromosomes (MACs) display several advantages as gene delivery vectors, such as stable episomal maintenance that avoids insertional mutations and the ability to carry large gene inserts including the regulatory elements. Previously, we showed that a MAC vector developed from a natural mouse chromosome by chromosome engineering was more stably maintained in adult tissues and hematopoietic cells in mice than HAC vectors. In this study, to expand the utility for a gene delivery vector in human cells and mice, we investigated the long-term stability of the MACs in cultured human cells and transchromosomic mice. We also investigated the chromosomal copy number-dependent expression of genes on the MACs in mice. The MAC was stably maintained in human HT1080 cells in vitro during long-term culture. The MAC was stably maintained at least to the F8 and F4 generations in ICR and C57BL/6 backgrounds, respectively. The MAC was also stably maintained in hematopoietic cells and tissues derived from old mice. Transchromosomic mice containing two or four copies of the MAC were generated by breeding. The DNA contents were comparable to the copy number of the MACs in each tissue examined, and the expression of the EGFP gene on the MAC was dependent on the chromosomal copy number. Therefore, the MAC vector may be useful not only for gene delivery in mammalian cells but also for animal transgenesis.


Assuntos
Instabilidade Cromossômica , Cromossomos Artificiais de Mamíferos/genética , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Animais , Linhagem Celular Tumoral , Feminino , Células Germinativas , Humanos , Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Fatores Sexuais
13.
Nat Rev Genet ; 13(1): 14-20, 2011 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-22179716

RESUMO

Mouse models have become an invaluable tool for understanding human health and disease owing to our ability to manipulate the mouse genome exquisitely. Recent progress in genomic analysis has led to an increase in the number and type of disease-causing mutations detected and has also highlighted the importance of non-coding regions. As a result, there is increasing interest in creating 'genomically' humanized mouse models, in which entire human genomic loci are transferred into the mouse genome. The technical challenges towards achieving this aim are large but are starting to be tackled with success.


Assuntos
Cromossomos Artificiais de Mamíferos/genética , Marcação de Genes/métodos , Técnicas de Transferência de Genes , Camundongos Transgênicos/genética , Transgenes/genética , Animais , Modelos Animais de Doenças , Doenças Genéticas Inatas/genética , Estudo de Associação Genômica Ampla/métodos , Humanos , Camundongos , Regiões Promotoras Genéticas , Recombinação Genética , Elementos Reguladores de Transcrição
14.
Methods Mol Biol ; 738: 101-10, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21431722

RESUMO

Recent technological advances have enabled visualization of the organization and dynamics of local -chromatin structures; however, the global mechanisms by which chromatin organization modulates gene regulation are poorly understood. We designed and constructed a human artificial chromosome (HAC) vector that allows regulation of transgene expression and delivery of a gene expression platform into many vertebrate cell lines. This technology for manipulating a transgene using a HAC vector could be used in applied biology.


Assuntos
Cromossomos Artificiais de Mamíferos/genética , Engenharia Genética/métodos , Vetores Genéticos/genética , Transgenes/genética , Animais , Células CHO , Fusão Celular , Cricetinae , Cricetulus , Expressão Gênica , Células HeLa , Humanos , Células Híbridas/citologia , Células Híbridas/metabolismo , Hibridização in Situ Fluorescente , Camundongos , Mitose , Mutagênese Insercional , Plasmídeos/genética , Transfecção
15.
Methods Mol Biol ; 738: 111-25, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21431723

RESUMO

Mammalian artificial chromosomes (MACs) are engineered chromosomes with defined genetic content that can function as non-integrating vectors with large carrying capacity and stability. The large carrying capacity allows the engineering of MACs with multiple copies of the same transgene, gene complexes, and to include regulatory elements necessary for the regulated expression of transgene(s). In recent years, different approaches have been explored to generate MACs (Vos Curr Opin Genet Dev 8:351-359, 1998; Danielle et al. Trends Biotech 23:573-583, 2005; Duncan and Hadlaczky Curr Opin Biotech 18:420-424, 2007): (1) the de novo formation by centromere seeding, the "bottom-up" approach, (2) the truncation of natural chromosomes or the modification of naturally occurring minichromosomes, the "top-down" approach, and (3) the in vivo "inductive" approach. Satellite DNA-based artificial chromosomes (SATACs) generated by the in vivo "inductive" method have the potential to become an efficient tool in diverse gene technology applications such as cellular protein manufacturing (Kennard et al. BioPharm Int 20:52-59, 2007; Kennard et al. Biotechnol Bioeng 104:526-539, 2009; Kennard et al. Biotechnol Bioeng 104:540-553, 2009), transgenic animal production (Telenius et al. Chromosome Res 7:3-7, 1999; Co et al. Chromosome Res 8:183-191, 2000; Monteith et al. Methods Mol Biol 240:227-242, 2003), and ultimately a safe vector for gene therapy (Vanderbyl et al. Stem Cells 22:324-333, 2004; Vanderbyl et al. Exp Hematol 33:1470-1476, 2005; Katona et al. Cell. Mol. Life Sci 65:3830-3838, 2008). A detailed protocol for the de novo generation of satellite DNA-based artificial chromosomes (SATACs) via induced large-scale amplification is presented.


Assuntos
Cromossomos Artificiais de Mamíferos/genética , DNA Satélite/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Animais , Fosfatos de Cálcio/química , Linhagem Celular , Precipitação Química , Células Clonais/citologia , Células Clonais/metabolismo , Cricetinae , Criopreservação , DNA Satélite/isolamento & purificação , Humanos , Desnaturação de Ácido Nucleico , Hibridização de Ácido Nucleico , Ribonucleases/metabolismo , Transfecção
16.
Methods Mol Biol ; 738: 127-40, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21431724

RESUMO

Advances in mammalian artificial chromosome technology have made chromosome-based vector technology amenable to a variety of biotechnology applications including cellular protein production, genomics, and animal transgenesis. A pivotal aspect of this technology is the ability to generate artificial chromosomes de novo, transfer them to a variety of cells, and perform downstream engineering of artificial chromosomes in a tractable and rational manner. Previously, we have described an alternative artificial chromosome technology termed the ACE chromosome system, where the ACE platform chromosome contains a multitude of site-specific, recombination sites incorporated during the creation of the ACE platform chromosome. In this chapter we review a variant of the ACE chromosome technology whereby site-specific, recombination sites can be integrated into the ACE chromosome following its de novo synthesis. This variation allows insertion of user-defined, site-specific, recombination systems into an existing ACE platform chromosome. These bioengineered ACE platform chromosomes, containing user-defined recombination sites, represent an ideal circuit board to which an array of genetic factors can be plugged-in and expressed for various research and therapeutic applications.


Assuntos
Cromossomos Artificiais de Mamíferos/genética , Mutagênese Insercional/métodos , Mutagênese Sítio-Dirigida/métodos , Recombinação Genética/genética , Animais , Linhagem Celular , Citometria de Fluxo , Humanos , Hibridização in Situ Fluorescente , Camundongos , Reprodutibilidade dos Testes
17.
Methods Mol Biol ; 738: 141-9, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21431725

RESUMO

Mammalian satellite DNA-based artificial chromosomes (SATACs) are unique among the mammalian artificial chromosomes. These reproducibly generated de novo chromosomes are stably maintained in different species, readily purified from the host cell's chromosomes and can be introduced into a variety of recipient cells. An artificial chromosome expression system (ACE system) has been developed on these SATACs to extend them for chromosome engineering. This system includes a Platform ACE containing multiple acceptor sites, specially designed targeting vector (ATV), and an ACE-integrase expression vector (pCXLamIntROK). Gene of interest are cloned into targeting vector (ATV), and site-specific loading of genes onto Platform ACE is facilitated by ACE-integrase mediated recombination. ACE system is suitable for multiple or subsequent loading of useful genes onto the same chromosome vector. This chapter describes the detailed procedure of chromosome engineering using the ACE system.


Assuntos
Bacteriófago lambda/enzimologia , Cromossomos Artificiais de Mamíferos/genética , Engenharia Genética/métodos , Integrases/metabolismo , Recombinação Genética , Animais , Células CHO , Cricetinae , Cricetulus , DNA/genética , DNA/isolamento & purificação , Camundongos , Plasmídeos/genética , Transformação Genética
18.
Methods Mol Biol ; 738: 151-60, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21431726

RESUMO

Gene therapy encounters important problems such as insertional mutagenesis caused by the integration of viral vectors. These problems could be circumvented by the use of mammalian artificial chromosomes (MACs) that are unique and high capacity gene delivery tools. MACs were delivered into various target cell lines including stem cells by microcell-mediated chromosome transfer (MMCT), microinjection, and cationic lipid and dendrimer mediated transfers. MACs were also cleansed to more than 95% purity before transfer with an expensive technology. We present here a method by which MACs can be delivered into murine embryonic stem (ES) cells with a nonexpensive, less tedious, but still efficient way.


Assuntos
Cromossomos Artificiais de Mamíferos/genética , Cromossomos Artificiais de Mamíferos/metabolismo , Dendrímeros/metabolismo , Técnicas de Transferência de Genes , Engenharia Genética , Animais , Células CHO , Células Clonais , Cricetinae , Cricetulus , Resistência a Medicamentos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Humanos , Cariotipagem , Mitose
19.
Methods Mol Biol ; 738: 161-81, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21431727

RESUMO

Horizontal gene transfer or simply transgenic technology has evolved much since 1980. Gene delivery strategies, systems, and equipments have become more and more precise and efficient. It has also been shown that even chromosomes can be used besides traditional plasmid and viral vectors for zygote or embryonic stem cell transformation. Artificial chromosomes and their loadable variants have brought their advantages over traditional genetic information carriers into the field of transgenesis. Engineered chromosomes are appealing vectors for gene transfer since they have large transgene carrying capacity, they are non-integrating, and stably expressing in eukaryotic cells. Embryonic stem cell lines can be established that carry engineered chromosomes and ultimately used in transgenic mouse chimera creation. The demonstrated protocol describes all the steps necessary for the successful production of transgenic mouse chimeras with engineered chromosome bearer embryonic stem cells.


Assuntos
Quimera/genética , Cromossomos Artificiais de Mamíferos/genética , Engenharia Genética , Camundongos Transgênicos/genética , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Cruzamento , Transferência Embrionária , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Técnicas de Transferência de Genes , Injeções , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mórula/citologia , Mórula/metabolismo , Agulhas , Transformação Genética , Zona Pelúcida/metabolismo
20.
Methods Mol Biol ; 738: 183-98, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21431728

RESUMO

Current transgenic technologies for gene transfer into the germline of mammals cause a random integration of exogenous naked DNA into the host genome that can generate undesirable position effects as well as insertional mutations. The vectors used to generate transgenic animals are limited by the amount of foreign DNA they can carry. Mammalian artificial chromosomes have large DNA-carrying capacity and ability to replicate in parallel with, but without integration into, the host genome. Hence they are attractive vectors for transgenesis, cellular protein production, and gene therapy applications as well. ES cells mediated chromosome transfer by conventional blastocyst injection has a limitation in unpredictable germline transmission. The demonstrated protocol of laser-assisted microinjection of artificial chromosome containing ES cells into eight-cell mouse embryos protocol described here can solve the problem for faster production of germline transchromosomic mice.


Assuntos
Cromossomos Artificiais de Mamíferos/genética , Técnicas de Transferência de Genes , Engenharia Genética , Lasers , Transplante de Células-Tronco , Células-Tronco/metabolismo , Animais , Cruzamento , Linhagem Celular , Células Clonais/citologia , Células Clonais/metabolismo , Transferência Embrionária , Feminino , Injeções , Masculino , Camundongos , Camundongos Transgênicos , Agulhas , Transformação Genética
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