人工染色体研究进展

人工染色体是人工构建的含有天然染色体基本功能单位的载体系统总称。人工染色体是非常优良的载体,具有超大的接受外源片段能力。由于不用整合到宿主基因组中,因此不会引起宿主基因的插入失活,及抑制转基因表达的位置效应。人工染色体已经从最初的酵母人工染色体(Yeast artificial chromosome,YAC)发展到细菌人工染色体(Bacterial artificial chromosome,BAC),再扩展到人类人工染色体(Human artificial chromosome,HAC)和植物人工染色体(Plant artificial chromosome,PAC)。文章就这4种人工染色体,尤其是植物人工染色体的研究进展和应用局限进行综述。目前,YAC和BAC已经广泛应用于基因组图谱制作、序列测定和基因克隆;HAC和PAC在基因治疗、外源医用蛋白的生产、新型优质高产高抗转基因作物构建中显现出广阔的应用前景。随着合成生物学的高速发展,美国科学家报道合成了一个"人造生命"。但是,和人工染色体一样,所谓的"人造生命",都是应用最新的基因工程技术,将不同的生命基础元件拼接组装而成,脱离了细胞环境并不能够自由存在。     

Artificial and engineered chromosomes: developments and prospects for gene therapy

At the gene therapy session of the ICCXV Chromosome Conference (2004), recent advances in the construction of engineered chromosomes and de novo human artificial chromosomes were presented. The long-term aims of these studies are to develop vectors as tools for studying genome and chromosome function and for delivering genes into cells for therapeutic applications. There are two primary advantages of chromosome-based vector systems over most conventional vectors for gene delivery. First, the transferred DNA can be stably maintained without the risks associated with insertion, and second, large DNA segments encompassing genes and their regulatory elements can be introduced, leading to more reliable transgene expression. There is clearly a need for safe and effective gene transfer vectors to correct genetic defects. Among the topics discussed at the gene therapy session and the main focus of this review are requirements for de novo human artificial chromosome formation, assembly of chromatin on de novo human artificial chromosomes, advances in vector construction, and chromosome transfer to cells and animals.     

In vivo modification of a maize engineered minichromosome

Engineered minichromosomes provide efficient platforms for stacking transgenes in crop plants. Methods for modifying these chromosomes in vivo are essential for the development of customizable systems for the removal of selection genes or other sequences and for the addition of new genes. Previous studies have demonstrated that Cre, a site-specific recombinase, could be used to modify lox sites on transgenes on maize minichromosomes; however, these studies demonstrated somatic recombination only, and modified minichromosomes could not be recovered. We describe the recovery of an engineered chromosome composed of little more than a centromere plus transgene that was derived by telomere-mediated truncation. We used the fiber fluorescence in situ hybridization technique and detected a transgene on the minichromosome inserted among stretches of CentC centromere repeats, and this insertion was large enough to suggest a tandem insertion. By crossing the minichromosome to a plant expressing Cre-recombinase, the Bar selection gene was removed, leaving behind a single loxP site. This study demonstrates that engineered chromosomes can be modified in vivo using site-specific recombinases, a demonstration essential to the development of amendable chromosome platforms in plants.     

Engineered chromosome regions with altered sequence composition demonstrate hierarchical large-scale folding within metaphase chromosomes

Mitotic chromosome structure and DNA sequence requirements for normal chromosomal condensation remain unknown. We engineered labeled chromosome regions with altered scaffold-associated region (SAR) sequence composition as a formal test of the radial loop and other chromosome models. Chinese hamster ovary cells were isolated containing high density insertions of a transgene containing lac operator repeats and a dihydrofolate reductase gene, with or without flanking SAR sequences. Lac repressor staining provided high resolution labeling with good preservation of chromosome ultrastructure. No evidence emerged for differential targeting of SAR sequences to a chromosome axis within native chromosomes. SAR sequences distributed uniformly throughout the native chromosome cross section and chromosome regions containing a high density of SAR transgene insertions showed normal diameter and folding. Ultrastructural analysis of two different transgene insertion sites, both spanning less than the full chromatin width, clearly contradicted predictions of simple radial loop models while providing strong support for hierarchical models of chromosome architecture. Specifically, an approximately 250-nm-diam folding subunit was visualized directly within fully condensed metaphase chromosomes. Our results contradict predictions of simple radial loop models and provide the first unambiguous demonstration of a hierarchical folding subunit above the level of the 30-nm fiber within normally condensed metaphase chromosomes.     

Expression of a reporter gene after microinjection of mammalian artificial chromosomes into pronuclei of bovine zygotes.

The introduction of mammalian artificial chromosomes (ACs) into zygotes represents an alternative, more predictive technology for the production of recombinant proteins in transgenic animals. The aim of these experiments was to examine the effects of artificial chromosome microinjection into bovine pronuclei on embryo development and reporter gene expression. Bovine oocytes aspirated from 2-5 mm size follicles were matured in vitro for 22 hr. Mature oocytes were fertilized in vitro with frozen- thawed bull spermatozoa. Artificial chromosome carrying either beta-galactosidase (Lac-Z) gene or green fluorescence protein (GFP) gene were isolated by flow cytometry. A single chromosome was microinjected into one of the two pronuclei of bovine zygotes. Sham injected zygotes served as controls. Injected zygotes were cultured in G 1.2 medium for 7 days. Hatched blastocysts were cultured on blocked STO cell feeder layer for attachment and outgrowth of ICM and trophectoderm cells. The results showed a high zygote survival rate following LacZ-ACs microinjection (74%). However, the blastocyst development rate after 7 days of culture was significantly lower than that of sham injected zygotes (7.5 vs. 22%). Embryonic cells positive for Lac-Z gene were detected by PCR in three of nine outgrowth colonies. In addition, GFP gene expression was observed in 15 out of 85 (18%) embryos at the arrested 2-cell stage to blastocyst stage. Six blastocysts successfully outgrew, three outgrowths were GFP positive for up to 3 weeks in culture. We conclude that the methodology for artificial chromosome delivery into bovine zygotes could lead to viable blastocyst development, and reporter gene expression could be sustained during pre-implantation development.     

Isotype switching of an immunoglobulin heavy chain transgene occurs by DNA recombination between different chromosomes

Transgenic mice carrying an immunoglobulin mu heavy chain transgene exhibit isotype switching of the transgene. We have now characterized the mechanism of transgene switching in these mice. The site of mu transgene insertion in one transgenic line has been localized to chromosome 5 using a series of polymorphic endogenous retroviruses as genetic markers in backcross mice. The endogenous immunoglobulin heavy chain locus resides on mouse chromosome 12, which shows that transgene isotype switching can occur between two different chromosomes even though normal antibody gene switching has generally been thought to occur within one chromosome. We find that transgene isotype switching involves interchromosomal DNA recombination, and our data suggest that the same enzymatic mechanisms mediate both normal isotype switch recombination and interchromosomal transgene switching. Our findings also support the notion that the isotype switching mechanism can induce chromosomal translocations such as observed for the c-myc gene in some B cell tumors.     

Engineering of polyploid Saccharomyces cerevisiae for secretion of large amounts of fungal glucoamylase

We engineered Saccharomyces cerevisiae cells that produce large amounts of fungal glucoamylase (GAI) from Aspergillus awamori var. kawachi. To do this, we used the delta-sequence-mediated integration vector system and the heat-induced endomitotic diploidization method. delta-Sequence-mediated integration is known to occur mainly in a particular chromosome, and the copy number of the integration is variable. In order to construct transformants carrying the GAI gene on several chromosomes, haploid cells carrying the GAI gene on different chromosomes were crossed with each other. The cells were then allowed to form spores, which was followed by dissection. Haploid cells containing GAI genes on multiple chromosomes were obtained in this way. One such haploid cell contained the GAI gene on five chromosomes and exhibited the highest GAI activity (5.93 U/ml), which was about sixfold higher than the activity of a cell containing one gene on a single chromosome. Furthermore, we performed heat-induced endomitotic diploidization for haploid transformants to obtain polyploid mater cells carrying multiple GAI genes. The copy number of the GAI gene increased in proportion to the ploidy level, and larger amounts of GAI were secreted.     

A structurally defined mini-chromosome vector for the mouse germ line

Yeast artificial mini-chromosomes have helped to define the features of chromosome architecture important for accurate segregation and replication and have been used to identify genes important for chromosome stability and as large-fragment cloning vectors. Artificial chromosomes have been developed in human cells but they do not have defined, experimentally predictable structures. Fragments of human chromosomes have also been introduced into mice and in one case passed through the germ line. In these experiments, however, the structure and sequence organization of the fragments was not defined. Structurally defined mammalian mini-chromosome vectors should allow large tracts of DNA to be introduced into the vertebrate germ line for biotechnological purposes and for investigations of features of chromosome structure that influence gene expression. Here, we have determined the structure and sequence organization of an engineered mammalian mini-chromosome, ST1, and shown that it is stably maintained in vertebrate somatic cells and that it can be transmitted through the mouse germ line.     

Engineering of Polyploid Saccharomyces cerevisiae for Secretion of Large Amounts of Fungal Glucoamylase

We engineered Saccharomyces cerevisiae cells that produce large amounts of fungal glucoamylase (GAI) from Aspergillus awamori var. kawachi. To do this, we used the δ-sequence-mediated integration vector system and the heat-induced endomitotic diploidization method. δ-Sequence-mediated integration is known to occur mainly in a particular chromosome, and the copy number of the integration is variable. In order to construct transformants carrying the GAI gene on several chromosomes, haploid cells carrying the GAI gene on different chromosomes were crossed with each other. The cells were then allowed to form spores, which was followed by dissection. Haploid cells containing GAI genes on multiple chromosomes were obtained in this way. One such haploid cell contained the GAI gene on five chromosomes and exhibited the highest GAI activity (5.93 U/ml), which was about sixfold higher than the activity of a cell containing one gene on a single chromosome. Furthermore, we performed heat-induced endomitotic diploidization for haploid transformants to obtain polyploid mater cells carrying multiple GAI genes. The copy number of the GAI gene increased in proportion to the ploidy level, and larger amounts of GAI were secreted.     

An X-linked human collagen transgene escapes X inactivation in a subset of cells.

Transgenic mice carrying one complete copy of the human alpha 1(I) collagen gene on the X chromosome (HucII mice) were used to study the effect of X inactivation on transgene expression. By chromosomal in situ hybridization, the transgene was mapped to the D/E region close to the Xce locus, which is the controlling element. Quantitative RNA analyses indicated that transgene expression in homozygous and heterozygous females was about 125% and 62%, respectively, of the level found in hemizygous males. Also, females with Searle's translocation carrying the transgene on the inactive X chromosome (Xi) expressed about 18% transgene RNA when compared to hemizygous males. These results were consistent with the transgene being subject to but partially escaping from X inactivation. Two lines of evidence indicated that the transgene escaped X inactivation or was reactivated in a small subset of cells rather than being expressed at a lower level from the Xi in all cells, (i) None of nine single cell clones carrying the transgene on the Xi transcribed transgene RNA. In these clones the transgene was highly methylated in contrast to clones carrying the transgene on the Xa. (ii) In situ hybridization to RNA of cultured cells revealed that about 3% of uncloned cells with the transgene on the Xi expressed transgene RNA at a level comparable to that on the Xa. Our results indicate that the autosomal human collagen gene integrated on the mouse X chromosome is susceptible to X inactivation. Inactivation is, however, not complete as a subset of cells carrying the transgene on Xi expresses the transgene at a level comparable to that when carried on Xa.     

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.     

Modeling chromosomes in mouse to explore the function of genes, genomic disorders, and chromosomal organization

One of the challenges of genomic research after the completion of the human genome project is to assign a function to all the genes and to understand their interactions and organizations. Among the various techniques, the emergence of chromosome engineering tools with the aim to manipulate large genomic regions in the mouse model offers a powerful way to accelerate the discovery of gene functions and provides more mouse models to study normal and pathological developmental processes associated with aneuploidy. The combination of gene targeting in ES cells, recombinase technology, and other techniques makes it possible to generate new chromosomes carrying specific and defined deletions, duplications, inversions, and translocations that are accelerating functional analysis. This review presents the current status of chromosome engineering techniques and discusses the different applications as well as the implication of these new techniques in future research to better understand the function of chromosomal organization and structures.     

Cloning and expression of transgenes using linear vectors in Trypanosoma cruzi

The identification of new targets for vaccine and drug development for the treatment of Chagas’ disease is dependent on deepening our understanding of the parasite genome. Vectors for genetic manipulation in Trypanosoma cruzi basically include those that remain as circular episomes and those that integrate into the parasite’s genome. Artificial chromosomes are alternative vectors to overcome problematic transgene expression often occurring with conventional vectors in this parasite. We have constructed a series of vectors named pTACs (Trypanosome Artificial Chromosomes), all of them carrying telomeric and subtelomeric sequences and genes conferring resistance to different selection drugs. In addition, one pTAC harbours a modified GFP gene (pTAC-gfp), and another one carries the ornithine decarboxilase gene from Crithidia fasciculata (pTAC-odc). We have encountered artificial chromosomes generated from pTACs in transformed T. cruzi epimastigotes for every version of the designed vectors. These extragenomic elements, in approximately 6–8 copies per cell, remained as linear episomes, contained telomeres and persisted after 150 and 60 generations with or without selection drugs, respectively. The linear molecules remained stable through the different T. cruzi developmental forms. Furthermore, derived artificial chromosomes from pTAC-odc could complement the auxotrophy of T. cruzi for polyamines. Our results show that pTACs constitute useful tools for reverse functional genetics in T. cruzi that will contribute to a better understanding of T. cruzi biology.     

Dual renin gene targeting by Cre-mediated interchromosomal recombination

This study describes a new approach to targeting clustered genes. Our study began with the establishment of two lines of mice carrying different mutations in either Ren1 or Ren2. These two genes, both encoding renin, span over 40 kb in tandem on chromosome 1. Each gene was mutated by gene targeting to contain loxP sites. These two mutants and Cre transgenic mice were mated to produce offspring carrying the mutant Ren1 and Ren2 genes, as well as the Cre transgene concurrently. Initially, two mutant Ren genes were located on separate chromosomes. Southern analysis of mice from the second generation revealed that the mutant Ren1 and Ren2 were interchromosomally recombined at the loxP sites to produce a new dually mutated allele on the chromosome at the rate of 9.6% (7/73). Thus, interchromosomal recombination can be efficiently programmed by mating as designed using the Cre-loxP system.     

Towards the development of better crops by genetic transformation using engineered plant chromosomes

Plant Biotechnology involves manipulation of genetic material to develop better crops. Keeping in view the challenges being faced by humanity in terms of shortage of food and other resources, we need to continuously upgrade the genomic technologies and fine tune the existing methods. For efficient genetic transformation, Agrobacterium-mediated as well as direct delivery methods have been used successfully. However, these methods suffer from many disadvantages especially in terms of transfer of large genes, gene complexes and gene silencing. To overcome these problems, recently, some efforts have been made to develop genetic transformation systems based on engineered plant chromosomes called minichromosomes or plant artificial chromosomes. Two approaches namely, “top-down” or “bottom-up” have been used for minichromosomes. The former involves engineering of the existing chromosomes within a cell and the latter de novo assembling of chromosomes from the basic constituents. While some success has been achieved using these chromosomes as vectors for genetic transformation in maize, however, more studies are needed to extend this technology to crop plants. The present review attempts to trace the genesis of minichromosomes and discusses their potential of development into plant artificial chromosome vectors. The use of these vectors in genetic transformation will greatly ameliorate the food problem and help to achieve the UN Millennium development goals.     

Artificial and engineered chromosomes: non-integrating vectors for gene therapy

Non-integrating gene-delivery platforms demonstrate promise as potentially ideal gene-therapy vector systems. Although several approaches are under development, there is little consensus as to what constitutes a true 'artificial' versus an 'engineered' human chromosome. Recent progress must be evaluated in light of significant technical challenges that remain before such vectors achieve clinical utility. Here, we examine the principal classes of non-integrating vectors, ranging from episomes to engineered mini-chromosomes to true human artificial chromosomes. We compare their potential as practical gene-transfer platforms and summarize recent advances towards eventual applications in gene therapy. Although chromosome-engineering technology has advanced considerably within recent years, difficulties in establishing composition of matter and effective vector delivery currently prevent artificial or engineered chromosomes being accepted as viable gene-delivery platforms.     

YAC transgenesis in farm animals: Rescue of albinism in rabbits

The generation of transgenic mice with mammalian genes cloned in yeast artificial chromosomes (YACs) has generated great interest in the field of gene transfer into livestock. Many of the problems associated with standard transgenesis—such as lack of crucial regulator elements and position effects related to the integration site, which lead to variation in expression levels irrespective of the dose of the transgene—have been practically overcome. The large size of YAC-derived gene constructs (in excess of 1 Mb) facilitates the presence and transfer of all elements required for the faithful regulation of a gene. With the experiments discussed in this report, we have addressed the possibility of applying the obvious advantages of YAC transgenesis to farm animals. We have generated transgenic rabbits carrying a 250 kb YAC covering the mouse tyrosinase gene by pronuclear microinjection, and thus rescued the albino phenotype of the transgenic individuals. To date, this is the first demonstration of a successful transfer of large genetic units into the germ line of farm animals. This development might improve the occurrence of transgene expression at physiological levels and specific sites in livestock. YAC transgenesis therefore will be applied in genetic engineering, for example, in the production of pharmacologically interesting proteins encoded by large gene units and generating transgenic donors for xenotransplantation. © 1996 Wiley-Liss, Inc.     

Construction of rice mini-chromosomes by telomere-mediated chromosomal truncation

Telomere truncation has been shown to be an efficient technology for the creation of mini-chromosomes that can be used as artificial chromosome platforms for genetic engineering. Artificial chromosome-based genetic engineering is considered to be superior to the existing techniques of randomized gene integration by Agrobacterium or biolistic-mediated genetic transformation. It organizes multiple transgenes as a unique genetic linkage block for subsequent manipulations in breeding. Telomere truncation technology relies on three components: the telomere sequence that mediates chromosomal truncation, a selection marker that allows the selection of transgenic events, and a site-specific recombination system that can be used to accept future genes into the mini-chromosome by gene targeting. These elements are usually pre-assembled before transformation, a process that is both time and labor consuming. We found in this research that the three elements could be mixed to transform plant cells in a biolistic transformation, and produced efficient chromosomal truncations and mini-chromosomes in rice. This system will allow rapid construction of mini-chromosomes with a flexible selection of resistant markers, site-specific recombination systems and other desirable elements. In addition, a rice telotrisomic line was used as the starting material for chromosomal truncations. Mini-chromosomes from the truncations of both the telocentric chromosome and other chromosomes were recovered. The mini-chromosomes remained stable during 2 years of subculture. The construction of mini-chromosomes in rice, an economically important crop, will provide a platform for future artificial chromosome-based genetic engineering of rice for stacking multiple genes.