Human Artificial Chromosome Vector Research Articles

Human artificial chromosome carrying 3p21.3-p22.2 region suppresses hTERT transcription in oral cancer cells

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.

Assembly of Multiple Full-Size Genes or Genomic DNA Fragments on Human Artificial Chromosomes Using the Iterative Integration System.

Human artificial chromosomes (HACs) are gene delivery vectors that have been used for decades for gene functional studies. HACs have several advantages over viral‐based gene transfer systems, including stable episomal maintenance in a single copy in the cell and the ability to carry up to megabase‐sized genomic DNA segments. We have previously developed the alphoidtetO‐HAC, which has a single gene acceptor loxP site that allows insertion of an individual gene of interest using Chinese hamster ovary (CHO) hybrid cells. The HAC, along with a DNA segment of interest, can then be transferred from donor CHO cells to various recipient cells of interest via microcell‐mediated chromosome transfer (MMCT). Here, we detail a protocol for loading multiple genomic DNA segments or genes into the alphoidtetO‐HAC vector using an iterative integration system (IIS) that utilizes recombinases Cre, ΦC31, and ΦBT. This IIS‐alphoidtetO‐HAC can be used for either serially assembling genomic loci or fragments of a large gene, or for inserting multiple genes into the same artificial chromosome. The insertions are executed iteratively, whereby each round results in the insertion of a new DNA segment of interest. This is accompanied by changes of expression of marker fluorescent proteins, which simplifies screening of correct clones, and changes of selection and counterselection markers, which constitutes an error‐proofing mechanism that removes mis‐incorporated DNA segments. In addition, the IIS‐alphoidtetO‐HAC carrying the genes can be eliminated from the cells, offering the possibility to compare the phenotypes of human cells with and without functional copies of the genes of interest. The resulting HAC molecules may be used to investigate biomedically relevant pathways or the regulation of multiple genes, and to potentially engineer synthetic chromosomes with a specific set of genes of interest. The IIS‐alphoidtetO‐HAC system is expected to be beneficial in creating multiple‐gene humanized models with the purpose of understanding complex multi‐gene genetic disorders. Published 2021. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Integration of the first DNA segment of interest into the IIS‐alphoidteto‐HAC Basic Protocol 2: Integration of a second DNA segment of interest into the IIS‐alphoidteto‐HAC Basic Protocol 3: Integration of a third DNA segment of interest into the IIS‐alphoidteto‐HAC Support Protocol: Fluorescence in situ hybridization analysis for the circular IIS‐alphoidtetO‐HAC

Construction of stable mouse artificial chromosome from native mouse chromosome 10 for generation of transchromosomic mice

Mammalian artificial chromosomes derived from native chromosomes have been applied to biomedical research and development by generating cell sources and transchromosomic (Tc) animals. Human artificial chromosome (HAC) is a precedent chromosomal vector which achieved generation of valuable humanized animal models for fully human antibody production and human pharmacokinetics. While humanized Tc animals created by HAC vector have attained significant contributions, there was a potential issue to be addressed regarding stability in mouse tissues, especially highly proliferating hematopoietic cells. Mouse artificial chromosome (MAC) vectors derived from native mouse chromosome 11 demonstrated improved stability, and they were utilized for humanized Tc mouse production as a standard vector. In mouse, however, stability of MAC vector derived from native mouse chromosome other than mouse chromosome 11 remains to be evaluated. To clarify the potential of mouse centromeres in the additional chromosomes, we constructed a new MAC vector from native mouse chromosome 10 to evaluate the stability in Tc mice. The new MAC vector was transmitted through germline and stably maintained in the mouse tissues without any apparent abnormalities. Through this study, the potential of additional mouse centromere was demonstrated for Tc mouse production, and new MAC is expected to be used for various applications.

Highly Efficient Microcell-Mediated Transfer of HACs Containing a Genomic Region of Interest into Mammalian Cells.

Human artificial chromosomes (HACs) are considered promising tools for gene delivery, functional analyses, and gene therapy. HACs have the potential to overcome many of the problems caused by the use of viral-based gene transfer systems, such as limited cloning capacity, lack of copy number control, and insertional mutagenesis during integration into host chromosomes. The recently developed alphoidtetO -HAC has an advantage over other HAC vectors because it can be easily eliminated from dividing cells by inactivation of its conditional kinetochore. This provides a unique control mechanism to study phenotypes induced by a gene or genes carried on the HAC. The alphoidtetO -HAC has a single gene acceptor loxP site that allows insertion of an individual gene of interest or a cluster of genes of up to several Mb in size in Chinese hamster ovary (CHO) hybrid cells. The HACs carrying chromosomal copies of genes can then be transferred from these donor CHO cells to different recipient cells of interest via microcell-mediated chromosome transfer (MMCT). Here, we describe a detailed protocol for loading a gene of interest into the alphoidtetO -HAC vector and for the subsequent transfer of the HAC to recipient cells using an improved MMCT protocol. The original MMCT protocol includes treatment of donor cells with colcemid to induce micronucleation, wherein the HAC becomes surrounded with a nuclear membrane. That step is followed by disarrangement of the actin cytoskeleton using cytochalasin B to help induce microcell formation. The updated MMCT protocol, described here, features the replacement of colcemid and cytochalasin B with TN16 + griseofulvin and latrunculin B, respectively, and the use of collagen/laminin surface coating to promote attachment of metaphase cells to plates during micronuclei induction. These modifications increase the efficiency of HAC transfer to recipient cells ten fold. The improved MMCT protocol has been successfully tested on several recipient cell lines, including human mesenchymal stem cells and mouse embryonic stem cells. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Insertion of a BAC containing a gene of interest into a single loxP loading site of alphoidtetO -HAC in hamster CHO cells Basic Protocol 2: Microcell-mediated chromosome transfer from donor hamster CHO cells to mammalian cells.

Abstract 1837: A human artificial chromosome (HAC)-based system as a powerful tool for stable co-expression of multiple proteins and therapeutic IgGs in mammalian cells

Abstract Background: Currently, there is increasing demand for recombinant therapeutic proteins produced in mammalian cell lines, especially therapeutic antibodies. MUC16-directed IgG and bispecific diabodies are one such example that represents a promising strategy for treatment of ovarian cancer. HEK293 and CHO cells are mostly used for large-scale stable expression of therapeutic proteins, however low protein yield, transgene silencing, cell line instability, and the time-consuming process of cell line development represent serious challenges. Here, we are presenting an alternative system for stable, high yield expression of multiple genes including complex therapeutic antibodies (HC and LC genes) and other proteins within single HAC. HAC vector propagate independently in cells without integration in the host chromosomes and has unlimited cloning capacity. Furthermore, this particular HAC can be targeted by transcriptional transactivators like tetR-VP16, eliminating transgene silencing, thereby providing stable long-term high yield protein production in mammalian cells. Methods: The targeting vector containing single or multiple genes is loaded at the unique LoxP site of tetO-HAC propagated in HPRT-deficient CHO cells by Cre-LoxP mediated recombination. As a result of recombination, functional HPRT gene will be reconstituted and HPRT-positive colonies are selected after 3 weeks growth in HAT medium. In the next step, HAC with therapeutic transgene(s) is transferred to the different high productive cell lines via MMCT technique following by high yield protein(s) expression in spinner flask. The alphoid tetO-HAC carries Bsr gene, so Blasticidin is used to select for the recipient cells that have taken up the HAC after Microcell-mediated chromosome transfer (MMCT). Results: Targeting vector with His-tagged MUC16 BiTEs gene (or iBAC caring HC and LC of anti-MUC16 IgG) was inserted into the HAC propagated in CHO cells. HPRT gene reconstitution was confirmed by PCR using a set of specific primers. Expression of His-tagged MUC16 BiTEs or anti-MUC16 IgG in CHO cells was confirmed by western blot and SDS-PAGE analysis. The yield of Ni- purified MUC16 BiTEs was up to 6 mg/l in original CHO cells. Our data from cellular models confirmed high efficacy of Ni- purified MUC16 BiTEs against OVCAR3 tumor cells, - up to 80% of the tumor cells were killed by T-cells in the presence of MUC16 BiTEs protein. After that, HAC/MUC16 BiTEs was successfully transferred to high productive ExpiHEK293F cells by MMCT. Therapeutic HAC transferring was confirmed by expression DsRed transgene linked with MUC16BiTEs through IRES and FISH analysis. FISH reveals the HAC propagated independently in ExpiHEK293F cells. Conclusions: Our data shows that the HAC is the new promising system for stable, high yield production of multiple therapeutic proteins. Citation Format: Artem Kononenko, Ian Laster, Oladapo Yeku, David Spriggs. A human artificial chromosome (HAC)-based system as a powerful tool for stable co-expression of multiple proteins and therapeutic IgGs in mammalian cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1837.

Engineering of human induced pluripotent stem cells via human artificial chromosome vectors for cell therapy and disease modeling

Genetic engineering of induced pluripotent stem cells (iPSCs) holds great promise for gene and cell therapy as well as drug discovery. However, there are potential concerns regarding the safety and control of gene expression using conventional vectors such as viruses and plasmids. Although human artificial chromosome (HAC) vectors have several advantages as a gene delivery vector, including stable episomal maintenance and the ability to carry large gene inserts, the full potential of HAC transfer into iPSCs still needs to be explored. Here, we provide evidence of a HAC transfer into human iPSCs by microcell-mediated chromosome transfer via measles virus envelope proteins for various applications, including gene and cell therapy, establishment of versatile human iPSCs capable of gene loading and differentiation into T cells, and disease modeling for aneuploidy syndrome. Thus, engineering of human iPSCs via desired HAC vectors is expected to be widely applied in biomedical research.

Enhancing the Therapeutic Efficacy of Bone Marrow-Derived Mononuclear Cells with Growth Factor-Expressing Mesenchymal Stem Cells for ALS in Mice.

SummarySeveral treatments have been attempted in amyotrophic lateral sclerosis (ALS) animal models and patients. Recently, transplantation of bone marrow-derived mononuclear cells (MNCs) was investigated as a regenerative therapy for ALS, but satisfactory treatments remain to be established. To develop an effective treatment, we focused on mesenchymal stem cells (MSCs) expressing hepatocyte growth factor, glial cell line-derived neurotrophic factor, and insulin-like growth factor using human artificial chromosome vector (HAC-MSCs). Here, we demonstrated the transplantation of MNCs with HAC-MSCs in ALS mice. As per our results, the progression of motor dysfunction was significantly delayed, and their survival was prolonged dramatically. Additional analysis revealed preservation of motor neurons, suppression of gliosis, engraftment of numerous MNCs, and elevated chemotaxis-related cytokines in the spinal cord of treated mice. Therefore, growth factor-expressing MSCs enhance the therapeutic effects of bone marrow-derived MNCs for ALS and have a high potential as a novel cell therapy for patients with ALS.

Multigene human artificial chromosome vector delivery with herpes simplex virus 1 amplicons

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.

An efficient protein production system via gene amplification on a human artificial chromosome and the chromosome transfer to CHO cells

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.

Transfer of Synthetic Human Chromosome into Human Induced Pluripotent Stem Cells for Biomedical Applications.

AlphoidtetO-type human artificial chromosome (HAC) has been recently synthetized as a novel class of gene delivery vectors for induced pluripotent stem cell (iPSC)-based tissue replacement therapeutic approach. This HAC vector was designed to deliver copies of genes into patients with genetic diseases caused by the loss of a particular gene function. The alphoidtetO-HAC vector has been successfully transferred into murine embryonic stem cells (ESCs) and maintained stably as an independent chromosome during the proliferation and differentiation of these cells. Human ESCs and iPSCs have significant differences in culturing conditions and pluripotency state in comparison with the murine naïve-type ESCs and iPSCs. To date, transferring alphoidtetO-HAC vector into human iPSCs (hiPSCs) remains a challenging task. In this study, we performed the microcell-mediated chromosome transfer (MMCT) of alphoidtetO-HAC expressing the green fluorescent protein into newly generated hiPSCs. We used a recently modified MMCT method that employs an envelope protein of amphotropic murine leukemia virus as a targeting cell fusion agent. Our data provide evidence that a totally artificial vector, alphoidtetO-HAC, can be transferred and maintained in human iPSCs as an independent autonomous chromosome without affecting pluripotent properties of the cells. These data also open new perspectives for implementing alphoidtetO-HAC as a gene therapy tool in future biomedical applications.

Improving the efficiency of gene insertion in a human artificial chromosome vector and its transfer in human-induced pluripotent stem cells.

A human artificial chromosome (HAC) vector has potential to overcome the problems of stable gene expression associated with plasmid, transposon, and virus-based vectors, such as insertional mutagenesis, position effect, uncontrollable copy number, unstable gene expression, and DNA size limitation. The main advantages of the HAC are its episomal nature and ability to accommodate DNA inserts of any size. However, HAC vectors have two disadvantages: low efficiency of gene insertion and lack of reports regarding the successful HAC transfer to human-induced pluripotent stem cells (iPSCs). We here provide the first report of a method for the efficient transfer of HAC to human iPSCs for obtaining reproducible experimental results. Moreover, we achieved a 10% increase in the gene insertion efficiency in the HAC vector using our new site-specific recombination systems VCre/VloxP and SCre/SloxP.

P.400 - Stunning pharmacological properties of DS-5141b, an antisense oligonucleotide consisting of 2'-O,4'-C-ethylene-bridged nucleic acids and 2'-O-methyl RNA, on dystrophin mRNA exon skipping

DS-5141b is an antisense oligonucleotide (ASO) consisting of 2'-O,4'-C-ethylene-bridged nucleic acids (ENA) and 2'-O-methyl RNA that induces dystrophin mRNA exon 45 skipping for treatment of Duchenne muscular dystrophy (DMD). As sufficient efficacy and safety had been observed in previous non-clinical studies of DS-5141b, DS-5141b currently has been in Ph1/2 clinical trial in Japan. In a repeated dosing study once a week for 4 weeks, DS-5141b showed clear exon 45 skipping at doses over 3 mg/kg in all examined tissues, anterior tibial muscles, diaphragm, and heart in mdx mice. It was remarkable that the exon 45 skipping in heart was detected at such a low dose. Based on the results and the past reports, we are persuaded that DS-5141b is the most potent exon skipping compound among ASOs in mdx mice. On the other hand, a phosphorodiamidate morpholino oligomer (PMO) with the same nucleotide sequence as that of DS-5141b did not induce the exon skipping in the all examined tissues at doses up to 30 mg/kg. 2'-O-methyl RNA with the same nucleotide sequence as that of DS-5141b showed much weaker exon skipping at 30 mg/kg compared to DS-5141b. Therefore, it was suggested that the stunning pharmacological properties of DS-5141b could be caused mainly by ENA. One of the most possible reasons of this differentiation is that ASOs containing ENA show extremely higher affinity to complementary mRNA than PMOs and 2'-O-methyl RNAs. However, the mechanisms responsible for the potent activity of DS-5141b are not fully understood. In order to make it clear in human dystrophin gene, now we are comparing effectiveness of exon 51 skipping of ASOs consisting of ENA and 2'-O-methyl RNA to eteplirsen, recently approved for DMD treatment by FDA, using mice expressing human dystrophin by a human artificial chromosome vector.

Effects of duration of electric pulse on in vitro development of cloned cat embryos with human artificial chromosome vector.

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 3.0kV/cm for either 30 or 60μs. 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 60μs. 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.

Development of Caco-2 cells co-expressing CYP3A4 and NADPH-cytochrome P450 reductase using a human artificial chromosome for the prediction of intestinal extraction ratio of CYP3A4 substrates

The Caco-2 cells co-expressing cytochrome P450 (CYP) 3A4 and NADPH-cytochrome P450 reductase (CPR) were developed using a human artificial chromosome (HAC) vector. The CYP3A4 and CPR genes were cloned into the HAC vector in CHO cells using the Cre-loxP system, and the microcell-mediated chromosome transfer technique was used to transfer the CYP3A4-CPR-HAC vector to Caco-2 cells. After seeding onto semipermeable culture inserts, the CYP3A4-CPR-HAC/Caco-2 cells were found to form tight monolayers, similar to the parental cells, as demonstrated by the high transepithelial electrical resistance (TEER) value and comparable permeability of non-CYP3A4 substrates between parent and CYP3A4-CPR-HAC/Caco-2 cell monolayers. The metabolic activity of CYP3A4 (midazolam 1′-hydroxylase activity) in the CYP3A4-CPR-HAC/Caco-2 cells was constant from 22 to 35 passages, indicating that HAC vectors conferred sufficient and sustained CYP3A4 activity to CYP3A4-CPR-HAC/Caco-2 cells. The strong relationship between the metabolic extraction ratios (ER) obtained from the CYP3A4-CPR-HAC/Caco-2 cells and calculated intestinal extraction ratios in humans (Eg) from reported intestinal availability (Fg) was found for 17 substrates of CYP3A4 (r2 = 0.84). The present study suggests that the CYP3A4-CPR-HAC/Caco-2 cell monolayer can serve as an in vitro tool that facilitates the prediction of intestinal extraction ratio (or availability) in humans.

Species-Specific Chromosome Engineering Greatly Improves Fully Human Polyclonal Antibody Production Profile in Cattle.

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.

Retargeting of microcell fusion towards recipient cell-oriented transfer of human artificial chromosome.

BackgroundHuman 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.ResultsHere, 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.ConclusionsRetargeted 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0142-z) contains supplementary material, which is available to authorized users.

Generation of a conditionally self-eliminating HAC gene delivery vector through incorporation of a tTAVP64 expression cassette

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 alphoidtetO-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 alphoidtetO-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 tTAVP64 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 alphoidtetO-HAC for routine gene function studies, we constructed a new TAR-BRV- tTAVP64 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 alphoidtetO-HAC in hamster CHO cells from where the HAC may be MMCT-transferred to the recipient human cells.

A pathway from chromosome transfer to engineering resulting in human and mouse artificial chromosomes for a variety of applications to bio-medical challenges.

Microcell-mediated chromosome transfer (MMCT) is a technique to transfer a chromosome from defined donor cells into recipient cells and to manipulate chromosomes as gene delivery vectors and open a new avenue in somatic cell genetics. However, it is difficult to uncover the function of a single specific gene via the transfer of an entire chromosome or fragment, because each chromosome or fragment contains a set of numerous genes. Thus, alternative tools are human artificial chromosome (HAC) and mouse artificial chromosome (MAC) vectors, which can carry a gene or genes of interest. HACs/MACs have been generated mainly by either a “top-down approach” (engineered creation) or a “bottom-up approach” (de novo creation). HACs/MACs with one or more acceptor sites exhibit several characteristics required by an ideal gene delivery vector, including stable episomal maintenance and the capacity to carry large genomic loci plus their regulatory elements, thus allowing the physiological regulation of the introduced gene in a manner similar to that of native chromosomes. The MMCT technique is also applied for manipulating HACs and MACs in donor cells and delivering them to recipient cells. This review describes the lessons learned and prospects identified from studies on the construction of HACs and MACs, and their ability to drive exogenous gene expression in cultured cells and transgenic animals via MMCT. New avenues for a variety of applications to bio-medical challenges are also proposed.

Developing de novo human artificial chromosomes in embryonic stem cells using HSV-1 amplicon technology.

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.

Generating a transgenic mouse line stably expressing human MHC surface antigen from a HAC carrying multiple genomic BACs.

The human artificial chromosome (HAC) vector is a promising tool to improve the problematic suppression and position effects of transgene expression frequently seen in transgenic cells and animals produced by conventional plasmid or viral vectors. We generated transgenic mice maintaining a single HAC vector carrying two genomic bacterial artificial chromosomes (BACs) from human HLA-DR loci (DRA and DRB1). Both transgenes on the HAC in transgenic mice exhibited tissue-specific expression in kidney, liver, lung, spleen, lymph node, bone marrow, and thymus cells in RT-PCR analysis. Stable functional expression of a cell surface HLA-DR marker from both transgenes, DRA and DRB1 on the HAC, was detected by flow cytometric analysis of splenocytes and maintained through at least eight filial generations. These results indicate that the de novo HAC system can allow us to manipulate multiple BAC transgenes with coordinated expression as a surface antigen through the generation of transgenic animals.Electronic supplementary materialThe online version of this article (doi:10.1007/s00412-014-0488-3) contains supplementary material, which is available to authorized users.