Human Artificial Chromosome Vector Research Articles

A novel expression system for genomic DNA loci using a human artificial chromosome vector with transformation-associated recombination cloning

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.

Construction of human artificial chromosome vectors by recombineering

Human artificial chromosomes (HACs) can be formed de novo by transfection of large fragments of cloned alphoid DNA into human HT1080 cells in tissue culture. In order to generate HACs carrying a gene of interest, one can either co-transfect the alphoid DNA and the gene of interest, or one can clone both into a single vector prior to transfection. Here we describe linking ∼70 kb of alphoid DNA onto a 156-kb BAC carrying the human HPRT gene using Red homologous recombination in the EL350 Escherichia coli host [Lee et al., Genomics 73 (2001) 56–65]. A selectable marker and EGFP marker were then added by loxP/Cre recombination using the arabinose inducible cre gene in the EL350 bacteria. The final construct generates minichromosomes in HT1080 cells and the HPRT gene is expressed. The retrofitting vector can be used to add the ∼70 kb of alphoid DNA to any BAC carrying a gene of interest to generate a HAC vector. The method can also be used to link any unrelated BAC or PAC insert onto another BAC clone. The EL350 bacteria are an excellent host for building up complex vectors by a combination of homologous and loxP/Cre recombination.

Human artificial chromosome (HAC) vector provides long-term therapeutic transgene expression in normal human primary fibroblasts

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.

Rapid creation of BAC-based human artificial chromosome vectors by transposition with synthetic alpha-satellite arrays

Efficient construction of BAC-based human artificial chromosomes (HACs) requires optimization of each key functional unit as well as development of techniques for the rapid and reliable manipulation of high-molecular weight BAC vectors. Here, we have created synthetic chromosome 17-derived alpha-satellite arrays, based on the 16-monomer repeat length typical of natural D17Z1 arrays, in which the consensus CENP-B box elements are either completely absent (0/16 monomers) or increased in density (16/16 monomers) compared to D17Z1 alpha-satellite (5/16 monomers). Using these vectors, we show that the presence of CENP-B box elements is a requirement for efficient de novo centromere formation and that increasing the density of CENP-B box elements may enhance the efficiency of de novo centromere formation. Furthermore, we have developed a novel, high-throughput methodology that permits the rapid conversion of any genomic BAC target into a HAC vector by transposon-mediated modification with synthetic alpha-satellite arrays and other key functional units. Taken together, these approaches offer the potential to significantly advance the utility of BAC-based HACs for functional annotation of the genome and for applications in gene transfer.

Construction of a novel human artificial chromosome vector for gene delivery

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.

991. Construction of a Novel Human Artificial Chromosome and Its Use for Gene Delivery

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.

Design and engineering human forms of monoclonal antibodies

AbstractThe antibody molecule has multiple properties that make it a key component of the immune response. These include its ability to recognize a vast array of different foreign substrates and to interact with and activate the host effector systems. Antibodies with defined specificities may serve as “magic bullets” for the diagnosis and therapy of multiple diseases. With the development of the hybridoma technology, it was possible to produce rodent (mouse or rat) monoclonal antibodies that are the product of a single clone of antibody producing cells and have only one antigen binding specificity. However, the therapeutic use of rodent monoclonals antibodies in humans is limited by their immunogenicity, short circulating half‐life, and inability to efficiently trigger human effector mechanisms. However, it proved difficult to produce human monoclonal antibodies using the same methods. To address these problems genetic engineering and expression systems have instead been used to produce chimeric, humanized, and totally human antibodies as well as antibodies with novel structures and functional properties. In addition, the use of yeast and human artificial chromosome vectors for animal transgenesis has allowed the development of animal models that produce antigen specific antibodies that are totally human. As a consequence, recombinant antibody‐based therapies are now used to treat a variety of clinical conditions including infectious diseases, inflammatory disorders, and cancer. This article summarizes and compares different strategies for designing and engineering human antibodies and their derivatives. Drug Dev. Res. 61:121–136, 2004. © 2004 Wiley‐Liss, Inc.

Human artificial chromosomes containing chromosome 17 alphoid DNA maintain an active centromere in murine cells but are not stable

Human artificial chromosomes (HACs) are autonomous molecules that can function and segregate as normal chromosomes in human cells. De novo HACs have successfully been used as gene expression vectors to complement genetic deficiencies in human cultured cells. HACs now offer the possibility of studying the regulation and expression of large genes in a variety of cell types from different tissues and correcting gene deficiencies caused by human inherited diseases. Complementary gene expression studies in mice, especially in mouse models of human genetic diseases, are also important in determining if large human transgenes can be expressed appropriately from artificial chromosomes. Toward this aim we are establishing artificial chromosomes in murine cells as novel gene expression vectors. Initially we transferred HAC vectors into murine cells, but were unable to generate de novo HACs at a reasonable frequency. We then transferred HACs previously established in human HT1080 cells to three different murine cell types by microcell fusion, followed by positive selection. We observed that the HACs in murine cells bound centromere protein C (CENP-C), a marker of active centromeres, and were detected under selection but rapidly lost when selection was removed. These results suggest that the HACs maintain at least a partially functional centromere complex in murine cells, but other factors are required for stability and segregation. Artificial chromosomes containing mouse centromeric sequences may be required for better stability and maintenance in murine cells.

Cloning of human centromeres by transformation-associated recombination in yeast and generation of functional human artificial chromosomes.

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.

Artificial chromosome vectors and expression of complex proteins in transgenic animals

Artificial chromosome vectors are autonomous, replicating DNA sequences containing a centromere, two telomeres and origins of replication. Artificial chromosomes have been proposed as possible vectors for transferring very large sequences of DNA into animals. Our goal has been to insert the entire human heavy- and light-chain immunoglobulin loci into cattle as a step in developing a production system for large quantities of human therapeutic polyclonal antibodies. A mitotically stable fragment of chromosome 14, containing the human heavy-chain locus, was identified. A chromosome cloning system was used to transfer the human lambda locus from an unstable chromosome 22 fragment to the chromosome 14 fragment to create a human artificial chromosome (HAC) carrying both immunoglobulin loci. The HAC vector was introduced into bovine primary fibroblasts. Selected fibroblast clones were rejuvenated and expanded by producing cloned fetuses. Cloned fetal cells were selected and recloned to produce 21 healthy, transchromosomic (Tc) calves. Four were analyzed and shown to functionally rearrange both heavy- and light-chain human immunoglobulin loci and produce human polyclonal antibodies. These results demonstrate the feasibility of using HAC vectors for production of transgenic livestock. More importantly, Tc cattle containing human immunoglobulin genes may be used to produce novel human polyclonal therapeutics.

Cloned transchromosomic calves producing human immunoglobulin.

Human polyclonal antibodies (hPABs) are useful therapeutics, but because they are available only from human donors, their supply and application is limited. To address this need, we prepared a human artificial chromosome (HAC) vector containing the entire unrearranged sequences of the human immunoglobulin (hIg) heavy-chain (H) and lambda (lambda) light-chain loci. The HAC vector was introduced into bovine primary fetal fibroblasts using a microcell-mediated chromosome transfer (MMCT) approach. Primary selection was carried out, and the cells were used to produce cloned bovine fetuses. Secondary selection was done on the regenerated fetal cell lines, which were then used to produce four healthy transchromosomic (Tc) calves. The HAC was retained at a high rate (78-100% of cells) in calves and the hIg loci underwent rearrangement and expressed diversified transcripts. Human immunoglobulin proteins were detected in the blood of newborn calves. The production of Tc calves is an important step in the development of a system for producing therapeutic hPABs.

Efficiency of de Novo Centromere Formation in Human Artificial Chromosomes

In a comparative study, we show that human artificial chromosome (HAC) vectors based on α-satellite (alphoid) DNA from chromosome 17 but not the Y chromosome regularly form HACs in HT1080 human cells. We constructed four structurally similar HAC vectors, two with chromosome 17 or Y alphoid DNA (17α, Yα) and two with 17α or Yα and the hypoxanthine guanine phosphoribosyltransferase locus (HPRT1). The 17α HAC vectors generated artificial minichromosomes in 32–79% of the HT1080 clones screened, compared with only ∼4% for the Yα HAC vectors, indicating that Yα is inefficient at forming a de novo centromere. The 17α HAC vectors produced megabase-sized, circular HACs containing multiple copies of alphoid fragments (60–250 kb) interspersed with either vector or HPRT1 DNA.The 17α-HPRT1 HACs were less stable than those with 17α only, and these results may influence the design of new HAC gene transfer vectors.

The Assembly of Large BACs by in Vivo Recombination

We have developed a method for recombining bacterial artificial chromosomes (BACs) and P1 artificial chromosomes (PACs) containing large genomic DNA fragments into a single vector using the Cre–lox recombination system from bacteriophage P1 in vivo. This overcomes the limitations of in vitro methods for generating large constructs based on restriction digestion, ligation, and transformation of DNA into Escherichia coli cells. We used the method to construct a human artificial chromosome vector of 404 kb encompassing long tracts of α satellite DNA, telomeric sequences, and the human hypoxanthine phosphoribosyltransferase gene. The specificity of Cre recombinase for loxP sites minimizes the possibility of intramolecular rearrangements, unlike previous techniques using general homologous recombination in E. coli, and makes our method compatible with the presence of large arrays of repeated sequences in cloned DNA. This methodology may also be applied to retrofitting PACs or BACs with markers and functional sequences.

Human artificial chromosomes generated by modification of a yeast artificial chromosome containing both human alpha satellite and single-copy DNA sequences.

A human artificial chromosome (HAC) vector was constructed from a 1-Mb yeast artificial chromosome (YAC) that was selected based on its size from among several YACs identified by screening a randomly chosen subset of the Centre d'Etude du Polymorphisme Humain (CEPH) (Paris) YAC library with a degenerate alpha satellite probe. This YAC, which also included non-alpha satellite DNA, was modified to contain human telomeric DNA and a putative origin of replication from the human beta-globin locus. The resultant HAC vector was introduced into human cells by lipid-mediated DNA transfection, and HACs were identified that bound the active kinetochore protein CENP-E and were mitotically stable in the absence of selection for at least 100 generations. Microdissected HACs used as fluorescence in situ hybridization probes localized to the HAC itself and not to the arms of any endogenous human chromosomes, suggesting that the HAC was not formed by telomere fragmentation. Our ability to manipulate the HAC vector by recombinant genetic methods should allow us to further define the elements necessary for mammalian chromosome function.