A mammalian artificial chromosome engineering system ace. In this work, we explore the efficacy of a bacterial artificial chromosome based vector applied to production of the constant region of the human igg1. Novel method to load a mammalian artificial chromosome. A mouse artificial chromosome mac vector can stably carry a large genomic region. The invention further provides a mac, wherein the unique cloning site is a nucleic acid sequence encoding a selectable marker. A mammalian chromosome is produced according to the method comprising the steps of introducing a dna construct comprising a mammalian telomere and a centromere into a mammalian cell, wherein the centromere has a dna sequence containing a plurality of copies of the cenpb box sequence consisting of. Targeted amplification of a sequence of interest in. Artificial chromosomes can be conveniently built and modified in yeast cells using. A mammalian artificial chromosome engineering system ace system applicable to biopharmaceutical protein production, transgenesis and genebased cell therapy.
Unique among macs, the mammalian satellite dnabased artificial chromosome. Cuttingedge and muchneeded, mammalian artificial chromosomes. The minimal length is unknown, but may be of several mb. Mammals, unlike budding yeast, depend on the histone h3 variant, cenpa, to epigenetically specify the location of the centromerethe locus essential for chromosome segregation.
A plasmid with a replication initiation region ir and a matrix attachment region mar initiates gene amplification in mammalian cells at a random chromosomal location. Common to the approaches taken was the inclusion of telomere repeats, which. Mammalian artificial chromosomes nature biotechnology. A method of producing a mammalian artificial chromosome of claim 23, wherein the mammalian cell is a mouse cell. An increased frequency of mammalian artificial chromosome formation was achieved through the modification of yac vectors to contain human centromeric sequences, from chromosome 21, and mammalian telomere arrays ikeno et al.
That is, instead of 46 chromosomes, the cell could have 47 with the 47th being very small, roughly 610 megabases mb in size instead of 50250 mb for natural chromosomes, and able to carry new genes introduced by human researchers. The resulting structure should be able to carry and express one or more selected genes transgenes, introduced for specific purposes. Principal investigator institute of genetics biological research center, hungarian academy of sciences phd. Methods and protocols offers a timely collection of meticulous techniques that will help elucidate the mechanisms of nuclear duplication, allow the introduction of changes into the human genome, and revolutionize our understanding of how chromosomes work. A method of producing a mammalian artificial chromosome of claim 23, wherein the dna construct is derived from saccharomyces cerevisiae. An efficient protein production system via gene amplification on a human artificial chromosome and the chromosome transfer to cho cells skip to. Stable gene expression from a mammalian artificial chromosome. Novel method to load a mammalian artificial chromosome mac with multiple genes ph.
To improve the retention rate of artificial chromosomes in mouse cells, we constructed novel mouse artificial chromosome mac vectors by truncating a natural mouse chromosome at a site adjacent to the centromeric region. Read this article to learn about the artificial chromosomes present in human genome. The present invention provides a mammalian artificial chromosome mac, comprising a centromere and a unique cloning site, said mac containing less than 0. The introduction of mammalian artificial chromosomes acs into zygotes represents an alternative, more predictive technology for the production of recombinant proteins in transgenic animals. A human artificial chromosome hac is a microchromosome that can act as a new chromosome in a population of human cells. By analogy with yeast artificial chromosomes, a mammalian artificial chromosome mac would be a molecule of defined structure comprising combinations of various cloned or synthesized chromosomal elements that had been individually manipulated, tested and optimized as part of a controlled construction process. A mammalian artificial chromosome engineering system ace system applicable to biopharmaceutical protein prod uction, transgenesis and genebased cell therapy. Brown oxford university, oxford, uk a mammalian artificial chromosome would enable analysis of the csacting dna sequences necessary for mammalian chromosome function and would allow large numbers of genes in a defined sequence environment to be introduced into experimental animals, agricultural livestock or human cells. Human artificial chromosomes that bypass centromeric dna. The design of a yac allows extremely large segments of genetic material to be inserted. Stable gene expression from a mammalian artificial. Yeast artificial chromosomes yacs are shuttlevectors that can be amplified in bacteria and employed for the cloning and manipulation of large deoxyribonucleic acid dna inserts up to 3 mb pairs in the yeast saccharomyces cerevisiae. To overcome these chromatin effects, we have employed a bacterial artificial chromosome bac as expression vector to obtain stable cell lines suitable for protein production. Bacterial artificial chromosomes improve recombinant.
Yeast artificial chromosomes yacs molecular biology. Methods and protocols, leading scientists in cell biology, genetics, cell culturing, and gene transfer present their most productive methods for the preparation, characterization, and use of artificial chromosomes. A mammalian artificial chromosome mac may be assembled through the juxtapposition of three kinds of dna elements. Artificial chromosome vectors also make it easier to store through bacterial cells rather than mammalian cells. Construction of an artificial molecule approximating to normal chromosome behaviour in a mammalian cell is an important first step which, until recently, has eluded us.
Artificial chromosome vectors artificial chromosomes are dna molecules assembled in vitro from defined constituents, which guarantee stable maintenance of large dna fragments with the properties of natural chromosomes. The aim of these experiments was to examine the effects of artificial chromosome microinjection into bovine pronuclei on embryo development and reporter gene expression. The centromeres are carried on stable chromosomes which carry no centromeres other than those comprising human dna sequences. Evaluation of an hprtluciferase reporter gene on a. Yeast artificial chromosome 1 yac the yeast artificial chromosome 2, which is often shortened to yac, is an artificially constructed system that can undergo replication. Rna interference rescue by bacterial artificial chromosome. Artificial chromosomes have long been touted as the ideal vector for gene therapy and biotechnology purposes based on the idea that such a chromosome would mimic the natural state of dna in the cell. Novel method to load multiple genes onto a mammalian. More recently, attempts are being made to construct artificial chromosomes in mammalian cells macs. Novel generation of human satellite dnabased artificial. Artificial chromosome an overview sciencedirect topics. This novel gene therapy method is called combined mammalian artificial chromosome stem cell therapy.
A mammalian chromosome is produced according to the method comprising the steps of. Mammalian artificial chromosomes are natural chromosomebased vectors that may carry a vast amount of genetic material in terms of both size and number. Pdf a mammalian artificial chromosome engineering system. The cell lines can be used to isolate the chromosomes as well as for use in inserting genes into mammalian cells. Mammalian artificial chromosome linkedin slideshare. They are reasonably stable and segregate well in both mitosis and meiosis.
School in biology the faculty of science and informatics university of szeged 2014. They are used to introduce and control new dna in a cell, to study how chromosomes function, and to map genes in genomes. A yeast artificial chromosome cloning vector consists of two copies of a yeast telomeric sequence telomeres are the sequences at the ends of chromosomes, a yeast centromere, a yeast ars an autonomously replicating sequence where dna replication begins, and. Mammalian artificial chromosomes and methods of using same. A mammalian artificial chromosome mac could be used for transferring genes into cells which could be beneficial for gene therapy and in the production of transgenic animals. Artificial chromosomes have now become a major research tool in both genome analysis and in the functional characterization of genes. In the remaining 5090% of metaphases in cell lines where a mac formed, the transfected dna either integrated into a host chromosome.
Purified mammalian aces can then be reintroduced into a variety of recipient cell lines where they have been stably. For gene therapy we need to have gene sized human dna fragments including their promoters and all the control elements. Formation of mammalian artificial chromosomes macs in cultured human cells following transfection of naked dna including candidate sequences considered essential for chromosome function has been reported by several groups harrington et al. We obtained cell clones containing the mac vectors that were stably maintained in mouse es cells and various tissues in tc mice. Recent breakthroughs with synthetic budding yeast chromosomes expedite the creation of synthetic mammalian chromosomes and genomes. The authors cover all relevant areas of artificial chromosome. Mammalian artificial chromosomes macs provide a means to introduce large payloads of genetic information into the cell in an autonomously replicating, nonintegrating format. Mammalian artificial chromosomes biologic research.
Mammalian artificial chromosomes methods and protocols. A novel and stable mouse artificial chromosome vector. Methods are taught for generating such cell lines from cell lines carrying dicentric chromosomes. An in vivo approach has been developed for generation of artificial chromosomes, based on the induction of intrinsic, largescale amplification mechanisms of mammalian cells. It is the potential application of artificial chromosomes is in gene therapy of human. Artificial chromosome formation rates and mac properties a fate of pac dna transfected intohprtdeficient ht1080 cells. Yeast artificial chromosomes yacs are shuttlevectors that can be amplified in bacteria and employed for the cloning and manipulation of large deoxyribonucleic acid dna inserts up to 3 mb. The gene transfer from one to another bacterial cell is called conjugation. The ace system, a mammalian artificial chromosome engineering technology. Bacterial artificial chromosome bac and yeast artificial chromosomes yac. Expression of a reporter gene after microinjection of. We propose a technology circumventing the problem of inefficient homologous recombination in mammalian cells by expressing an orthologous gene from a closely related species, including its regulatory sequences carried on a bacterial artificial chromosome bac. They cloned large fragments of human chromosomes into a mitotically stable human. This, it is argued, would mean that essentially unlimited amounts of dna could be incorporated into such a vector enabling either large genes or whole metabolic pathways to be.