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Microcell-mediated chromosome transfer (MMCT) technology enables a single and intact mammalian chromosome or megabase-sized chromosome fragments to be transferred from donor to recipient cells. The conventional MMCT method is performed immediately after the purification of microcells. The timing of the isolation of microcells and the preparation of recipient cells is very important. Thus, ready-made microcells can improve and simplify the process of MMCT. Here, we established a cryopreservation method to store microcells at −80 °C, and compared these cells with conventionally- (immediately-) prepared cells with respect to the efficiency of MMCT and the stability of a human artificial chromosome (HAC) transferred to human HT1080 cells. The HAC transfer in microcell hybrids was confirmed by FISH analysis. There was no significant difference between the two methods regarding chromosome transfer efficiency and the retention rate of HAC. Thus, cryopreservation of ready-to-use microcells provides an improved and simplified protocol for MMCT.

Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance, and the ability to carry large gene inserts. We previously developed HAC vectors from the normal human chromosomes using a chromosome engineering technique. However, endogenous genes were remained in these HACs, limiting their therapeutic applications. In this study, we refined a HAC vector without endogenous genes from human chromosome 21 in homologous recombination-proficient chicken DT40 cells. The HAC was physically characterized using a transformation-associated recombination (TAR) cloning strategy followed by sequencing of TAR-bacterial artificial chromosome clones. No endogenous genes were remained in the HAC. We demonstrated that any desired gene can be cloned into the HAC using the Cre-loxP system in Chinese hamster ovary cells, or a homologous recombination system in DT40 cells. The HAC can be efficiently transferred to other type of cells including mouse ES cells via microcell-mediated chromosome transfer. The transferred HAC was stably maintained in vitro and in vivo. Furthermore, tumor cells containing a HAC carrying the suicide gene, herpes simplex virus thymidine kinase (HSV-TK), were selectively killed by ganciclovir in vitro and in vivo. Thus, this novel HAC vector may be useful not only for gene and cell therapy, but also for animal transgenesis.

We previously reported that sirtuin 2 (SIRT2), a mammalian member of the NAD+-dependent protein deacetylases, participates in mitotic regulation, specifically, in efficient mitotic cell death caused by the spindle checkpoint. Here, we describe a novel function of SIRT2 that is different from mitotic regulation. SIRT2 down-regulation using siRNA caused apoptosis in cancer cell lines such as HeLa cells, but not in normal cells. The apoptosis was caused by p53 accumulation, which is mediated by p38 MAPK activation-dependent degradation of p300 and the subsequent MDM2 degradation. Sirtuin inhibitors are emerging as antitumor drugs, and this function has been ascribed to the inhibition of SIRT1, the most well-characterized sirtuin that deacetylases p53 to promote cell survival and also binds to other proteins in response to genotoxic stress. This study suggests that SIRT2 can be a novel molecular target for cancer therapy and provides a molecular basis for the efficacy of SIRT2 for future cancer therapy.

Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance that avoids insertional mutations and the ability to carry large gene inserts including regulatory elements. Multipotent germline stem (mGS) cells have a great potential for gene therapy because they can be generated from an individual's testes, and when reintroduced can contribute to the specialized function of any tissue. As a proof of concept, we herein report the functional restoration of a genetic deficiency in mouse p53-/- mGS cells, using a HAC with a genomic human p53 gene introduced via microcell-mediated chromosome transfer. The p53 phenotypes of gene regulation and radiation sensitivity were complemented by introducing the p53-HAC and the cells differentiated into several different tissue types in vivo and in vitro. Therefore, the combination of using mGS cells with HACs provides a new tool for gene and cell therapies. The next step is to demonstrate functional restoration using animal models for future gene therapy.

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.

The recent emergence of stem cell-based tissue engineering has now opened up new venues for gene therapy. The task now is to develop safe and effective vectors that can deliver therapeutic genes into specific stem cell lines and maintain long-term regulated expression of these genes. Human artificial chromosomes (HACs) possess several characteristics that require gene therapy vectors, including a stable episomal maintenance, and the capacity for large gene inserts. HACs can also carry genomic loci with regulatory elements, thus allowing for the expression of transgenes in a genetic environment similar to the chromosome. Currently, HACs are constructed by a two prone approaches. Using a top-down strategy, HACs can be generated from fragmenting endogenous chromosomes. By a bottom-up strategy, HACs can be created de novo from cloned chromosomal components using chromosome engineering. This review describes the current advances in developing HACs, with the main focus on their applications and potential value in gene delivery, such as HAC-mediated gene expression in embryonic, adult stem cells, and transgenic animals.

A number of gene delivery systems are currently being developed for potential use in gene therapy. Here, we demonstrate the feasibility of 21deltaqHAC, a newly developed human artificial chromosome (HAC), as a gene delivery system. We first introduced a 21deltaqHAC carrying an EGFP reporter gene and a geneticin-resistant gene (EGFP-21deltaqHAC) into hematopoietic cells by microcell-mediated chromosome transfer. These HAC-containing hematopoietic cells showed resistance to geneticin, expressed EGFP and retained the ability to differentiate into various lineages, and the EGFP-21deltaqHAC was successfully transduced into primary hematopoietic cells. Hematopoietic cells harboring the EGFP-21deltaqHAC could still be detected at two weeks post-transplantation in immunodeficient mice. We also showed effective expansion of hematopoietic cells by introducing the 21deltaqHAC containing ScFvg, a gp130-based chimeric receptor that transmits growth signals in response to specific-antigen of this receptor. All of these results demonstrate the usefulness of HAC in gene therapy.

Human centromeres remain poorly characterized regions of the human genome despite their importance for the maintenance of chromosomes. In part this is due to the difficulty of cloning of highly repetitive DNA fragments and distinguishing chromosome-specific clones in a genomic library. In this work we report the highly selective isolation of human centromeric DNA using transformation-associated recombination (TAR) cloning. A TAR vector with alphoid DNA monomers as targeting sequences was used to isolate large centromeric regions of human chromosomes 2, 5, 8, 11, 15, 19, 21 and 22 from human cells as well as monochromosomal hybrid cells. The alphoid DNA array was also isolated from the 12 Mb human mini-chromosome DeltaYq74 that contained the minimum amount of alphoid DNA required for proper chromosome segregation. Preliminary results of the structural analyses of different centromeres are reported in this paper. The ability of the cloned human centromeric regions to support human artificial chromosome (HAC) formation was assessed by transfection into human HT1080 cells. Centromeric clones from DeltaYq74 did not support the formation of HACs, indicating that the requirements for the existence of a functional centromere on an endogenous chromosome and those for forming a de novo centromere may be distinct. A construct with an alphoid DNA array from chromosome 22 with no detectable CENP-B motifs formed mitotically stable HACs in the absence of drug selection without detectable acquisition of host DNAs. In summary, our results demonstrated that TAR cloning is a useful tool for investigating human centromere organization and the structural requirements for formation of HAC vectors that might have a potential for therapeutic applications.

Genomic imprinting is the phenomenon by which the two alleles of certain genes are differentially expressed according to their parental origin. Extensive analysis of allelic expression at multiple imprinted loci in a normal population has not performed so far. In the present study, we examined the allelic expression pattern of three imprinted genes in a panel of 262 Japanese normal individuals. We observed differences in the extent of maintenance of allele-specific expression of the three genes. The allelic expression of small nuclear ribonucleoprotein N (SNRPN) was stringently regulated while that of multimembrane-spanning polyspecific transporter-like gene 1 (IMPT1) showed a large degree of variation. Significant biallelic expression of insulin-like growth factor II (IGF2) was observed in about 10% of normal individuals. Our findings add to the accumulating evidence for variable allelic expression at multiple loci in a normal human population. This epigenetic heterogeneity can be a stable trait and potentially influence individual phenotypes.

Chromosome fragments represent feasible gene delivery vectors with the use of microcell-mediated chromosome transfer. To test a prerequisite for a gene delivery vector, we examined the stability of human chromosome fragments (hCFs) in cultured cells and in trans-chromosomic (Tc) mice. Fragments of human chromosomes 2 (hCF(2-W23)), 11 (hCF-11) and 14 (hCF(SC20)) tagged with neo were introduced into the TT2F mouse ES cells, and retention of the hCFs was examined by FISH during long-term culture without selection. In contrast to the gradual loss of hCF(2-W23) and hCF-11, hCF(SC20) remained stable over 70 population doublings in the ES cells. The hCF(SC20) was also stable in cultured human tumor cells and chicken DT40 cells. We have previously generated chimeric mice using the ES cells harboring the hCF(2-W23) or hCF(SC20), followed by production of Tc mice. Although both the hCF(2-W23) and hCF(SC20) persisted in cells of Tc mice as an additional chromosome and were transmitted to offspring, the hCF(SC20) was more stable than the hCF(2-W23) in F1 and F2 mice. The present study shows that the stability of hCFs in Tc mice differs with tissue types and with genetic background used for successive breedings. Thus, the hCF(SC20), which was relatively stable in both mouse and human cells, may be a promising candidate for development as a gene delivery vector.

Human chromosome fragments and human artificial chromosomes (HAC) represent feasible gene delivery vectors via microcell-mediated chromosome transfer. Strategies to construct HAC involve either 'build up' or 'top-down' approaches. For each approach, techniques for manipulating HAC in donor cells in order to deliver HAC to recipient cells are required. The combination of chromosome fragments or HAC with microcell-mediated chromosome transfer has facilitated human gene mapping and various genetic studies. The recent emergence of stem cell-based tissue engineering has opened up new avenues for gene and cell therapies. The task now is to develop safe and effective vectors that can deliver therapeutic genes into specific stem cells and maintain long-term regulated expression of these genes. Although the transfer-efficiency needs to be improved, HAC possess several characteristics that are required for gene therapy vectors, including stable episomal maintenance and the capacity for large gene insets. HAC can also carry genomic loci with regulatory elements, which allow for the expression of transgenes in a genetic environment similar to the natural chromosome. This review describes the lessons and prospects learned, mainly from recent studies in developing HAC and HAC-mediated gene expression in embryonic and adult stem cells, and in transgenic animals.

We previously identified SIRT2, an nicotinamide adenine dinucleotide (NAD)-dependent tubulin deacetylase, as a protein downregulated in gliomas and glioma cell lines, which are characterized by aneuploidy. Other studies reported SIRT2 to be involved in mitotic progression in the normal cell cycle. We herein investigated whether SIRT2 functions in the mitotic checkpoint in response to mitotic stress caused by microtubule poisons. By monitoring chromosome condensation, the exogenously expressed SIRT2 was found to block the entry to chromosome condensation and subsequent hyperploid cell formation in glioma cell lines with a persistence of the cyclin B/cdc2 activity in response to mitotic stress. SIRT2 is thus a novel mitotic checkpoint protein that functions in the early metaphase to prevent chromosomal instability (CIN), characteristics previously reported for the CHFR protein. We further found that histone deacetylation, but not the aberrant DNA methylation of SIRT2 5'untranslated region is involved in the downregulation of SIRT2. Although SIRT2 is normally exclusively located in the cytoplasm, the rapid accumulation of SIRT2 in the nucleus was observed after treatment with a nuclear export inhibitor, leptomycin B and ionizing radiation in normal human fibroblasts, suggesting that nucleo-cytoplasmic shuttling regulates the SIRT2 function. Collectively, our results suggest that the further study of SIRT2 may thus provide new insights into the relationships among CIN, epigenetic regulation and tumorigenesis.

Following the recent completion of the human genome sequence, genomics research has shifted its focus to understanding gene complexity, expression, and regulation. However, in order to investigate such issues, there is a need to develop a practical system for genomic DNA expression. Transformation-associated recombination (TAR) cloning has proven to be a convenient tool for selective isolation of a genetic locus from a complex genome as a circular YAC using recombination in yeast. The human artificial chromosome (HAC) vector containing an acceptor loxP site has served as a platform for the reproducible expression of transgenes. In this study, we describe a system that efficiently expresses a genetic locus in mammalian cells by retrofitting a TAR-YAC with the donor loxP site and loading it onto the HAC vector by the Cre/loxP system. In order to demonstrate functional expression of genomic loci, the entire human hypoxanthine phosphoribosyl transferase (HPRT) locus contained in a 100 kb YAC was loaded onto the HAC vector and was shown to complement the genetic defect in Hprt-deficient CHO cells. Thus, the combination of TAR cloning and the HAC vector may serve as a powerful tool for functional genomic studies.

Human artificial chromosomes (HACs) segregating freely from host chromosomes are potentially useful to ensure both safety and duration of gene expression in therapeutic gene delivery. However, low transfer efficiency of intact HACs to the cells has hampered the studies using normal human primary cells, the major targets for ex vivo gene therapy. To elucidate the potential of HACs to be vectors for gene therapy, we studied the introduction of the HAC vector, which is reduced in size and devoid of most expressed genes, into normal primary human fibroblasts (hPFs) with microcell-mediated chromosome transfer (MMCT). We demonstrated the generation of cytogenetically normal hPFs harboring the structurally defined and extra HAC vector. This introduced HAC vector was retained stably in hPFs without translocation of the HAC on host chromosomes. We also achieved the long-term production of human erythropoietin for at least 12 weeks in them. These results revealed the ability of HACs as novel options to circumvent issues of conventional vectors for gene therapy.

Efficient regulation of transgene would greatly facilitate the analysis of gene function in biological systems for basic research and clinical applications. The tetracycline-regulatable system (TRS) has proven to be a promising tool for such purposes. Despite their widespread application, a number of challenges are still associated with the use of TRS, including clonal variability in the regulation and copy number. We have recently constructed a novel human artificial chromosome (HAC) called 21ΔqHAC. By housing a TRS-based DNA-PKcs expression cassette in this HAC, we were able to circumvent the problems associated with conventional TRS-based vectors. We achieved tight control of DNA-PKcs expression and rescued the radiosensitive phenotype of DNA-PKcs-deficient CHO cells. The combined use of HAC and the TRS serves as a model for controllable and fixed copy number expression vectors. Our study also demonstrates the suitability of the HAC to accommodate multi-subunit constructs such as that of the TRS.

Potential problems of conventional transgenes include insertional disruption of the host genome and unpredictable, irreproducible expression of the transgene by random integration. Alternatively, human artificial chromosomes (HACs) can circumvent some of the problems. Although several HACs were generated and their mitotic stability was assessed, a practical way for introducing exogenous genes by the HACs has yet to be explored. In this study, we developed a novel HAC from sequence-ready human chromosome 21 by telomere-directed chromosome truncation and added a loxP sequence for site-specific insertion of circular DNA by the Cre/loxP system. This 21HAC vector, delivered to a human cell line HT1080 by microcell fusion, bound centromere proteins A, B, and C and was mitotically stable during long-term culture without selection. The EGFP gene inserted in the HAC vector expressed persistently. These results suggest that the HAC vector provides useful system for functional studies of genes in isogenic cell lines.

Measurement of the relative amounts of transcripts from two alleles is important in the study of imprinted genes, since quantitative differences that vary among tissues or individuals, and subtle differences in the ratio of allelic expression can have pathobiological significance. Discrimination of alleles is commonly based on PCR, followed by restriction endonuclease digestion to recognize a polymorphic site. However, the use of restriction enzymes misses most of the available single nucleotide polymorphisms. Practically, it requires substantial post-PCR analyses including the restriction enzyme digestion and gel electrophoresis, all of which increase turn around time. Taking advantage of our previous study identifying lung adenocarcinomas displaying biallelic expression of the imprinted gene MEST, we investigated the validity of a method of allelic discrimination in a real-time PCR assay using allele-specific probes. Allelic expression of the MEST gene in the range of 4-fold differences was detected. This new method should enhance our ability to rapidly and accurately assess allelic expression of imprinted genes in a number of samples.

The introduction of a human chromosome 1 via microcell-mediated chromosome transfer (MMCT) induces the cellular senescence in mouse melanoma B16-F10 cells. The senescent cells maintained still the telomerase activity, which is frequently associated with immortal growth of human cells, suggesting that a telomerase-independent mechanism is involved in the senescence observed in this mouse cell line. To map the senescence-inducing gene to a specific chromosomal region, we took two experimental approaches: identification of a minimal region with the senescence-inducing activity via MMCT of a series of subchromosomal transferrable fragments (STFs), each consisting of a different profile of human chromosome 1-derived regions, and identification of a region commonly deleted from the transferred chromosome 1 in the revertant clones that escaped cellular senescence. These approaches identified a 2.7-3.0 Mb of senescence-inducing region shared among the active STFs and a 2.4-3.0 Mb of commonly deleted region in the revertant clones. These two regions overlapped each other to map the responsible gene at the 450 to 600-kb interval between UniSTS93710 and D1S3542 on chromosome 1q42.3. This study provides essential information and materials for cloning and characterization of a novel senescence-inducing gene that functions in a telomerase-independent pathway, which is likely to be conserved between mice and humans.

We generated transchromosomal (Tc) mice containing a human chromosome 21 fragment (hCF21) using mouse embryonic stem (ES) cells with the transferred hCF21. Here we report breeding analyses that test the maintenance rate of the hCF21 in Tc mice of two different genetic backgrounds, MCH (ICR) and C57BL/6. Fluorescence in situ hybridization and polymerase chain reaction-based DNA analyses revealed that the structure of the hCF21 fragment including the CBR1, SIM2, HLCS, and D21S268 markers, was approximately 5 Mb in size, and was transmitted at least to the F3 generation. Though the retention rate of the hCF21 was variable among individual mice, for example, 21%–92% in brain and 10%–92% in tail fibroblasts, the C57BL/6 background yielded a higher retention rate than did the MCH (ICR). These results suggest that the hCF21 could be maintained stably in Tc mice, depending on the genetic background. The panel of Tc mice will be a useful model to investigate the function of genes on the hCF21 fragment in various tissues through germinal transmission.

Genomic imprinting is a parental origin–specific chromosomal modification that causes differential expression of maternal and paternal alleles of a gene. Accumulating evidence suggests that deregulation of imprinted genes, including loss of imprinting (LOI), plays a role in oncogenesis. In the present study, we investigated allelic expression of six imprinted genes in human lung adenocarcinomas as well as in matched normal lung tissue. Informative cases showing heterozygosity for the gene of interest were selected from 35 patients. LOI of the insulin-like growth factor 2 gene (IGF2) and mesoderm-specific transcript (MEST, also known as paternally expressed gene 1) was noted in 47% (seven of 15) and 85% (11 of 13) of informative cases, respectively. Monoallelic expression was maintained in all the matched normal tissues examined. LOI of IGF2 was seen more frequently in moderately to poorly differentiated adenocarcinomas. In contrast, H19, small nuclear ribonucleoprotein–associated polypeptide N gene (SNRPN), necdin gene (NDN), and long QT intronic transcript 1 (LIT1) exhibited consistent monoallelic expression in all the informative samples. These findings indicated that independent deregulation took place in imprinted genes and suggested that aberrant imprinting of IGF2 and MEST was involved in the development of lung adenocarcinoma. © 2001 Wiley-Liss, Inc.

Trisomy 21 (Ts21) is the most common live-born human aneuploidy; it results in a constellation of features known as Down's syndrome (DS). Ts21 is the most frequent cause of congenital heart defects and the leading genetic cause of mental retardation. To investigate the gene dosage effects of an extra copy of human chromosome 21 (Chr 21) on various phenotypes, we used microcell-mediated chromosome transfer to create embryonic stem (ES) cells containing Chr 21. ES cell lines retaining Chr 21 as an independent chromosome were used to produce chimeric mice with a substantial contribution from Chr 21-containing cells. Fluorescence in situ hybridization and PCR-based DNA analysis revealed that Chr 21 was substationally intact but had sustained a small deletion. The freely segregating Chr 21 was lost during development in some tissues, resulting in a panel of chimeric mice with various mosaicism as regards retention of the Chr 21. These chimeric mice showed a high correlation between retention of Chr 21 in the brain and impairment in learning or emotional behavior by open-field, contextual fear conditioning and forced swim tests. Hypoplastic thymus and cardiac defects, i.e. double outlet right ventricle and riding aorta, were observed in a considerable number of chimeric mouse fetuses with a high contribution of Chr 21. These chimeric mice mimic a wide variety of phenotypic traits of DS, revealing the utility of mice containing Chr 21 as unique models for DS and for the identification of genes responsible for DS.

Mammalian imprinted genes are frequently arranged in clusters on particular chromosomes. The imprinting cluster on human chromosome 11p15 is associated with Beckwith-Wiedemann syndrome (BWS) and a variety of human cancers. To clarify the genomic organization of the imprinted cluster, an extensive screen for differentially expressed transcripts in the 11p15 region was performed using monochromosomal hybrids with a paternal or maternal human chromosome 11. Here we describe an imprinted antisense transcript identified within the KvLQT1 locus, which is associated with multiple balanced chromosomal rearrangements in BWS and an additional breakpoint in embryonal rhabdoid tumors. The transcript, called LIT1 (long QT intronic transcript 1), was expressed preferentially from the paternal allele and produced in most human tissues. Methylation analysis revealed that an intronic CpG island was specifically methylated on the silent maternal allele and that four of 13 BWS patients showed complete loss of maternal methylation at the CpG island, suggesting that antisense regulation is involved in the development of human disease. In addition, we found that eight of eight Wilms' tumors exhibited normal imprinting of LIT1 and five of five tumors displayed normal differential methylation at the intronic CpG island. This contrasts with five of six tumors showing loss of imprinting of IGF2. We conclude that the imprinted gene domain at the KvLQT1 locus is discordantly regulated in cancer from the imprinted domain at the IGF2 locus. Thus, this positional approach using human monochromosomal hybrids could contribute to the efficient identification of imprinted loci in humans.

To obtain cosmid markers and transcribed sequences from a specific chromosome region, a series of radiation-reduced hybrids (RHs) containing various regions of human chromosome 11 was prepared from microcell hybrid A9 (neo11) cells containing a normal human chromosome 11 tagged with pSV2neo at 11p11.2. Among 15 radiation hybrid clones isolated, RH(11)-9 which contains a q23 fragment in addition to theneo integration site, was used for the construction of a cosmid library. Cosmid clones having human DNA sequences were screened, and localized by Southern hybridization with the radiation hybrid panel. Fifty-nine cosmids were assigned to 11q23 and 6 cosmids to 11p11.2. Exon amplification proceeded with 23 of the 59 cosmids and 16 putative exons were cloned. Three of them were identical to those constituting a known gene which locates on q23 (ATDC), and the others were unknown. Thus, the RHs containing various subchromosomal fragments of chromosome 11 were useful for constructing region-specific DNA markers. The RH-(11)-9 cells and putative exons also facilitate the positional cloning of genes in the 11q23 region.

In the conventional virus and non-viral vectors for gene therapy, it comes up with crucial issues to be solved; 1) The introduced vector might develop lesions on the host chromosome and the fears for safety such as the malignant transformation are mentioned, 2) The copy number of the introduced gene is not regulated, 3) Sufficient expression in the host cell is not sustained for long term, 4) The introduced gene is inactivated or suffers the effects of controlled sequence on the host chromosome.

A group of genes on a human chromosome or its fragment (hCF) is transferrable from donor to recipient cells by microcell-mediated chromosome transfer. Since the hCF is autonomously replicated and segregated as an extra chromosome in the host cells, 1) it does not disrupt the genomic context of the host, 2) its copy number is restricted and persistent and 3) up to megabase- sized DNA fragment is transferable. Our previous study revealed that use of engineered hCFs successfully achieved delivery and functional expression of human immunoglobulin genes in mice. But carry over of genes out of interest on hCF is not suitable for therapeutic applications. In addition, chromosome engineering requires substantial steps and it is not practical to prepare hCF panels corresponding broad range of genes. We aimed at constructing a structurally defined human artificial chromosome (HAC) vector into which circular DNA can be inserted by Cre/lox system. We chose the human chromosome 21 (hChr21) for the HAC construction, since sequence information is available from public databases. The hChr21 was transferred to homologous recombination proficient chicken DT40 cells, followed by truncation of distal q-arm by telomere seeding, introduction of loxP sequence by targeted integration and finally truncation of distal q- arm. Removal of q- and p-arm was confirmed by PCR and FISH analysis. These engineered hCFs named 21HAC vectors were transferrable from DT40 to human HT1080 and mouse ES cells, via CHO cells. Long-term culture in the absence of selection revealed that the 21HAC vectors could stably maintained in the HT1080 and mouse ES cells. Site-specific insertion of circular DNA into the loxP site on the 21 HAC vector was tested using GFP gene expression cassette as a reporter in HT1080 cells. Correct insertion was selected by reconstruction of neo gene split on the HAC vector and a circular DNA insert, that confers G418 resistance. Persistent expression of the introduced GFP gene was observed during serial culture in medium without G418, indicating that the loxP site integrated in the pericentromeric region was not heterochromatinized. Mouse ES cells carrying the GFP/21HAC vector was induced neuronal differentiation in vitro. Colonies detected by antibody against neuron-specific -tubulin expressed GFP, suggesting that the presence of the HAC vector did not disturb differentiation of ES cells. These results indicated that the HAC vector can be used for functional study of transgene in human and mouse cells. The 21HAC vectors could be potentially developed as gene delivery vector applicable for ex vivo gene therapy. Prerequisite for a gene delivery vector is stable maintenance in somatic cells and performance of gene expression regulation. To address these issues, following studies are ongoing; 1) tissue specific gene expression in hepatocytes under regulation of albumin enhancer/promoter, 2) induction of tissue specific gene expression during in vitro differentiation of human Mesenchymal Stem Cells and 3) construction of non- cells in which insulin expression is inducible. Current progress in these studies will be reported.

We have recently constructed a novel HAC vector called 21ΔqHAC, generated by deletion of a distal portion of the long arm of human chromosome 21 and insertion of a loxP sequence to facilitate insertion of any desired gene construct. We intend to use this HAC vector to rescue T and B cell development and the radiosensitive phenotype in the DNA-PKcs-deficient SCID (severe combined immunodeficiency) mice. Our approach will involve introduction of 21ΔqHAC containing the DNA-PKcs gene into DNA-PKcs-deficient ES cells, differentiation of corrected ES cells into blood stem cells and transplantation of these blood stem cells into SCID mice. To evaluate the usefulness of 21ΔqHAC vector for this purpose, we first attempted in vitro studies using CHO cells (V3) exhibiting radiosensitive phenotype due to a DNA-PKcs deficiency. In this experiment, we inserted a ~21 kb doxycycline-inducible DNA-PKcs expression cassette into 21ΔqHAC vector by Cre-mediated recombination. The resulting 21ΔqHAC/DNA-PKcs construct was then introduced into the V3 cells. RT-PCR results from cells containing the 21ΔqHAC/DNA-PKcs vector showed doxycycline-dependent regulation of the DNA-PKcs gene. Furthermore, radiosensitivity assays showed significant phenotype rescue in these cells due to the production of functional DNA-PKcs protein. To our knowledge, our study is the first to report successful artificial control of gene expression by placing the multiple regulatory elements of the tetracycline system in a single expression cassette within a HAC vector. Our results also show that 21ΔqHAC is a very useful tool in the introduction of a therapeutic gene and the correction of a defective phenotype.

Combination of functional genomics approaches and microarray analyses to define candidate senescence genes