Current advances in microcell-mediated chromosome transfer technology and its applications.

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.

Correlation between luminescence intensity and cytotoxicity in cell-based cytotoxicity assay using luciferase.

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.

Mammalian Synthetic Biology: Time for Big MACs.

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.

Mouse embryonic stem cells with a multi-integrase mouse artificial chromosome for transchromosomic mouse generation.

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.

De novo formed satellite DNA-based mammalian artificial chromosomes and their possible applications.

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.

Novel method to load multiple genes onto a mammalian artificial chromosome.

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.

Highly stable maintenance of a mouse artificial chromosome in human cells and mice.

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.

Genomically humanized mice: technologies and promises.

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.

Construction and use of a bottom-up HAC vector for transgene expression.

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.

De novo generation of satellite DNA-based artificial chromosomes by induced large-scale amplification.

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.

Downstream bioengineering of ACE chromosomes for incorporation of site-specific recombination cassettes.

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.

Chromosome engineering with lambda-integrase mediated recombination system: the ACE system.

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.

Dendrimer mediated transfer of engineered chromosomes.

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.

Engineered chromosomes in transgenics.

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.

Transfer of stem cells carrying engineered chromosomes with XY clone laser system.

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.

Mammalian artificial chromosomes and clinical applications for genetic modification of stem cells: an overview.

Modifying multipotent, self-renewing human stem cells with mammalian artificial chromosomes (MACs), present a promising clinical strategy for numerous diseases, especially ex vivo cell therapies that can benefit from constitutive or overexpression of therapeutic gene(s). MACs are nonintegrating, autonomously replicating, with the capacity to carry large cDNA or genomic sequences, 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 progenitor cells. The status quo is that 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. We will describe the progress of MAC technologies, the subsequent modifications of stem cells, and discuss the establishment of MAC platform stem cell lines to facilitate proof-of-principle studies and preclinical development.

Engineered mammalian chromosomes in cellular protein production: future prospects.

The manufacture of recombinant proteins at industrially relevant levels requires technologies that can engineer stable, high expressing cell lines rapidly, reproducibly, and with relative ease. Commonly used methods incorporate transfection of mammalian cell lines with plasmid DNA containing the gene of interest. Identifying stable high expressing transfectants is normally laborious and time consuming. To improve this process, the use of engineered chromosomes has been considered. To date, the most successful technique has been based on the artificial chromosome expression or ACE System, which consists of the targeted transfection of cells containing mammalian based artificial chromosomes with multiple recombination acceptor sites. This ACE System allows for the specific transfection of single or multiple gene copies and eliminates the need for random integration into native host chromosomes. The utility of using artificial engineered mammalian chromosomes, specifically the ACE System, is illustrated in several case studies covering the generation of CHO cell lines expressing monoclonal antibodies.

[Efficient production of transfected human keratinocytes under serum-free and feeder layer-free conditions]. / Effiziente Herstellung transfizierter humaner Keratinozyten unter serum- und Feederlayer-freien Bedingungen.

PURPOSE/BACKGROUND: Keratinocyte transplantation after burn injury and in chronic wound treatment is a potentially useful method in clinical practice. As transfer of keratinocytes is easily monitored and gene expression is controllable by topical administration of inductors, keratinocyte cultures are an especially interesting medium for gene therapeutic approaches far above of wound healing applications. A major obstacle is the standardization of keratinocyte preparation and maintenance of pure proliferative cultures for clinical application. The best outcomes in previous protocols were obtained using fibroblasts as a feeder layer, a requirement for long-term expanded cultures. Cell expansion and a high purity of keratinocytes are prerequisites for clinical transfer studies. Here, we describe a human keratinocytes preparation method that allows cell proliferation and expansion in culture without a feeder layer. MATERIALS AND METHODS: Human keratinocytes were prepared from skin biopsies and cultured on untreated plastic culture dishes using Waymouth medium the first days followed by a change to a commercially available serum-free keratinocyte medium. The cells were characterized morphologically followed by transfection. For positive selection, transfected cells were selected by the cotransfection system pMACS Kk and magnetic cell sorting. RESULTS: Transfection rates were determined by expression of GFP vector which were 35%. The usage of magnetic cell sorting resulted in positive selection of transfected cells. Positive cells were able to adhere and proliferate after the sorting procedure. High viability and expansion of plastic adherent keratinocytes was achieved allowing up to 5 passages without signs of senescence and the doubling times were 3-5 days. The cells displayed typical keratinocyte morphology and immunostaining confirmed high keratinocyte purity. The number of contaminating fibroblasts was low. CONCLUSION: Here, we describe an efficient and inexpensive method for a standardized human keratinocyte isolation without the need of a fibroblast feeder layer. This protocol may facilitate the clinical application of cell based therapies in burn injuries or chronic wounds using keratinocytes.

A combined artificial chromosome-stem cell therapy method in a model experiment aimed at the treatment of Krabbe's disease in the Twitcher mouse.

Mammalian artificial chromosomes (MACs) are safe, stable, non-integrating genetic vectors with almost unlimited therapeutic transgene-carrying capacity. The combination of MAC and stem cell technologies offers a new strategy for stem cell-based therapy, the efficacy of which was confirmed and validated by using a mouse model of a devastating monogenic disease, galactocerebrosidase deficiency (Krabbe's disease). Therapeutic MACs were generated by sequence-specific loading of galactocerebrosidase transgenes into a platform MAC, and stable, pluripotent mouse embryonic stem cell lines were established with these chromosomes. The transgenic stem cells were thoroughly characterized and used to produce chimeric mice on the mutant genetic background. The lifespan of these chimeras was increased twofold, verifying the feasibility of the development of MAC-stem cell systems for the delivery of therapeutic genes in stem cells to treat genetic diseases and cancers, and to produce cell types for cell replacement therapies.