Chromosome Transfer Research Articles

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

Genome Mosaicism in Field Strains of Mycoplasma bovis as Footprints of In-Host Horizontal Chromosomal Transfer.

Horizontal gene transfer was long thought to be marginal in Mollicutes, but the capacity of some of these wall-less bacteria to exchange large chromosomal regions has been recently documented. Mycoplasma chromosomal transfer (MCT) is an unconventional mechanism that relies on the presence of a functional integrative conjugative element (ICE) in at least one partner and involves the horizontal acquisition of small and large chromosomal fragments from any part of the donor genome, which results in progenies composed of an infinite variety of mosaic genomes. The present study focuses on Mycoplasma bovis, an important pathogen of cattle responsible for major economic losses worldwide. By combining phylogenetic tree reconstructions and detailed comparative genome analyses of 36 isolates collected in Spain (2016 to 2018), we confirmed the mosaic nature of 16 field isolates and mapped chromosomal transfers exchanged between their hypothetical ancestors. This study provides evidence that MCT can take place in the field, most likely during coinfections by multiple strains. Because mobile genetic elements (MGEs) are classical contributors of genome plasticity, the presence of phages, insertion sequences (ISs), and ICEs was also investigated. Data revealed that these elements are widespread within the M. bovis species and evidenced classical horizontal transfer of phages and ICEs in addition to MCT. These events contribute to wide-genome diversity and reorganization within this species and may have a tremendous impact on diagnostic and disease control. IMPORTANCE Mycoplasma bovis is a major pathogen of cattle that has significant detrimental effects on economics and animal welfare in cattle rearing worldwide. Understanding the evolution and the adaptative potential of pathogenic mycoplasma species in the natural host is essential to combating them. In this study, we documented the occurrence of mycoplasma chromosomal transfer, an atypical mechanism of horizontal gene transfer, in field isolates of M. bovis that provide new insights into the evolution of this pathogenic species in their natural host. Although these events are expected to occur at low frequency, their impact is accountable for genome-wide variety and reorganization within M. bovis species, which may compromise both diagnostic and disease control.

IVF embryo choices and pregnancy outcomes.

Investigate the chromosome status and transfer outcomes of embryos selected using routine "best morphology" IVF practices. A prospective multi-center, non-selection cohort study involving patients undertaking IVF treatment. Study entry conditions were blastocyst biopsy, >1 embryo with chromosome analysis and frozen transfer of the best morphology embryo. Primary analyses were βhCG positive, implantation, ongoing pregnancy and birth rates and pregnancy-stage progression failures. After transfer, embryo chromosome status was assigned and outcomes divided into two primary groups - euploids (n=135) and aneuploids (n=53). Compared to euploid embryo transfers, aneuploid embryos had significantly lower primary outcomes (+βhCG: 67% vs. 30%, p<0.0001; IR: 56% vs. 19%, p<0.0001; ongoing week 12: 51% vs. 9%, p<0.0001; and livebirths: 50% vs. 8%, p<0.0001, respectively). Transfers were further subdivided into smaller groups according to their main chromosomal feature. Stage analysis showed higher failure rates for aneuploids to initiate a pregnancy (p<0.0001), higher subclinical miscarriage rate (p=0.0402) and higher clinical miscarriage rate (p=0.0038). Routine morphology-based embryo selection resulted in a high euploid selection rate but a significant number of aneuploid embryos were still inadvertently selected for transfer (28%) with the subsequent high failure rates for pregnancy initiation and progression having implications for appropriate patient management.

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.

Human chromosome 3p21.3 carries TERT transcriptional regulators in pancreatic cancer

Frequent loss of heterozygosity (LOH) on the short arm of human chromosome 3 (3p) region has been found in pancreatic cancer (PC), which suggests the likely presence of tumor suppressor genes in this region. However, the functional significance of LOH in this region in the development of PC has not been clearly defined. The human telomerase reverse transcriptase gene (hTERT) contributes to unlimited proliferative and tumorigenicity of malignant tumors. We previously demonstrated that hTERT expression was suppressed by the introduction of human chromosome 3 in several cancer cell lines. To examine the functional role of putative TERT suppressor genes on chromosome 3 in PC, we introduced an intact human chromosome 3 into the human PK9 and murine LTPA PC cell lines using microcell-mediated chromosome transfer. PK9 microcell hybrids with an introduced human chromosome 3 showed significant morphological changes and rapid growth arrest. Intriguingly, microcell hybrid clones of LTPA cells with an introduced human chromosome 3 (LTPA#3) showed suppression of mTert transcription, cell proliferation, and invasion compared with LTPA#4 cells containing human chromosome 4 and parental LTPA cells. Additionally, the promoter activity of mTert was downregulated in LTPA#3. Furthermore, we confirmed that TERT regulatory gene(s) are present in the 3p21.3 region by transfer of truncated chromosomes at arbitrary regions. These results provide important information on the functional significance of the LOH at 3p for development and progression of PC.

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.

Identification of Candidate Progenitor Populations Which Causes Transient Abnormal Myelopoiesis

Transient abnormal myelopoiesis (TAM) is a temporal preleukemic status specific to neonates of Down syndrome (DS). TAM is characterized by leucocytosis and/or thrombocytopenia with the trisomy of chromosome 21 and somatic GATA1 mutation. Morphologically, most blasts found in TAM are myeloblasts, and some other types of blasts are similar to those typically found in DS-related acute megakaryoblastic leukemia (DS-AMKL). Although TAM patients usually experience spontaneous remission within 6 months, 20 to 30 % of develop DS-AMKL. The cell population that causes TAM phenotypes in vitro or in vivo mouse and human models has not yet been determined.Recently, the hematopoietic differentiation systems from human pluripotent stem cells (PSCs) have revealed the pathogenesis in various diseases including TAM. Previous TAM models have succeeded in recapitulating the phenotypes, such as arrest and/or skewness of differentiation from immature hematopoietic progenitor cells (HPCs). However, identification of the responsible cell population for TAM at a precise subpopulation level based on functional evaluations has not yet been accomplished. Therefore, we applied our 2D-hematopoietic differentiation system for identifying the responsible cell population through the progenitor assay. A comprehensive understanding of TAM pathogenesis will enable us to predict and eventually prevent the disease progression from TAM into life-threatening DS-AMKL.First, we differentiated artificial trisomy 21 embryonic stem cells (Ts21-ESCs) generated by transfer of human artificial chromosome (HAC) 21 in our 2D-differentiation system. Five subpopulations of HPCs were harvested at day6 and day9, and cells were subjected to expression analysis of hematopoiesis-related genes. Principal component analysis (PCA) successfully mapped each HPC subpopulation corresponding to the hematopoietic developmental pathways, proving that our system can be used for isolating functional HPC subpopulations.Next, we used genome edition to establish strictly controlled isogenic ES/induced pluripotent stem cell (iPSC) pairs derived from TAM, non-TAM/DS patients and the Ts21-ESCs. Then we differentiated ES/iPSCs into each hematopoietic lineage to identify the abnormal phenotypes in vitro . GATA1 -mutated clones (G1s-clones) showed obviously different phenotypes, compared to GATA1-WT clones (WT-clones). In particular, completely restricted erythroid lineage cells (CD42b-CD71+CD235a+) and increased myeloid lineage cells (CD34-CD235a-CD41-CD42b-CD43+CD45+) were observed in G1s-clones. Moreover, in megakaryocytic lineage, the frequency of immature (CD235a-CD41+CD42bdim) and mature (CD235a-CD41+CD42bbright) cells differed between G1s- and WT-clones. Notably, the proportion of immature megakaryoblasts increased in G1s-clones. These data indicated that we have succeeded in recapitulation the TAM phenotype with our in vitro differentiation system.Then, we performed progenitor assay based on in vitro TAM phenotypes. We differentiated 5 isolated HPCs subpopulation into erythrocyte, megakaryocyte and myeloid cells with a suspension culture and colony forming assay. As a result, we found strong correlations between the increased or decreased specific cell fractions and abnormal phenotypes. Correlation pattern is slightly different between clones.In conclusion, we have identified putative responsible cell populations that contribute to the pathological phenotype in TAM. These populations were located on a series of spectra in the differentiation stage. Ubiquitous expression of GATA1 during the differentiation process and wide heterogeneity of the differentiation systems in general may be the reason why precise identification of the single responsible population was not possible. However, our model of TAM using iPSCs enabled us to narrow down the candidate cell populations which have the potential to be a source of DS-AMKL. The identification of responsible progenitor cells is an important first step to predict the transformation from TAM into DS-AMKL and to prevent its progression. Moreover, the method used in this study, such as 2D-differentiation system and progenitor assay, is a promising way to identify the responsible cell population of various types of leukemia other than TAM. DisclosuresNo relevant conflicts of interest to declare.

Refactoring the Conjugation Machinery of Promiscuous Plasmid RP4 into a Device for Conversion of Gram-Negative Isolates to Hfr Strains.

Chromosomal exchange and subsequent recombination of the cognate DNA between bacteria was one of the most useful genetic tools (e.g., Hfr strains) for genetic analyses of E. coli before the genomic era. In this paper, yeast assembly has been used to recruit the conjugation machinery of environmentally promiscuous RP4 plasmid into a minimized, synthetic construct that enables transfer of chromosomal segments between donor/recipient strains of P. putida KT2440 and potentially many other Gram-negative bacteria. The synthetic device features [i] a R6K suicidal plasmid backbone, [ii] a mini-Tn5 transposon vector, and [iii] the minimal set of genes necessary for active conjugation (RP4 Tra1 and Tra2 clusters) loaded as cargo in the mini-Tn5 mobile element. Upon insertion of the transposon in different genomic locations, the ability of P. putida-TRANS (transference of RP4-activated nucleotide segments) donor strains to mobilize genomic stretches of DNA into neighboring bacteria was tested. To this end, a P. putida double mutant ΔpyrF (uracil auxotroph) Δedd (unable to grow on glucose) was used as recipient in mating experiments, and the restoration of the pyrF+/edd+ phenotypes allowed for estimation of chromosomal transfer efficiency. Cells with the inserted transposon behaved in a manner similar to Hfr-like strains and were able to transfer up to 23% of their genome at frequencies close to 10–6 exconjugants per recipient cell. The hereby described TRANS device not only expands the molecular toolbox for P. putida, but it also enables a suite of genomic manipulations which were thus far only possible with domesticated laboratory strains and species.

The Pros and Cons of Rye Chromatin Introgression into Wheat Genome

Rye is one of the most commonly used sources of elite genes in wheat improvement programs. Due to the high collinearity of the genomes of both cereal species, it is possible to obtain interspecific chromosomal translocations and substitution lines. Rye chromatin is used to transfer numerous genes for resistance to biotic and abiotic stresses into the wheat genome. Introgression has also resulted in improved agronomic traits. However, despite the numerous advantages, the transfer of large fragments or whole chromosomes has been quite often accompanied by a decrease in end-use quality. This paper presents an overview of the benefits and drawbacks of using rye as a source of variability in wheat breeding.

Horizontal chromosome transfer and independent evolution drive diversification in Fusarium oxysporum f. sp. fragariae.

Summary The genes required for host‐specific pathogenicity in Fusarium oxysporum can be acquired through horizontal chromosome transfer (HCT). However, it is unknown if HCT commonly contributes to the diversification of pathotypes.Using comparative genomics and pathogenicity phenotyping, we explored the role of HCT in the evolution of F. oxysporum f. sp. fragariae, the cause of Fusarium wilt of strawberry, with isolates from four continents.We observed two distinct syndromes: one included chlorosis (‘yellows‐fragariae’) and the other did not (‘wilt‐fragariae’). All yellows‐fragariae isolates carried a predicted pathogenicity chromosome, ‘chrY‐frag’, that was horizontally transferred at least four times. chrY‐frag was associated with virulence on specific cultivars and encoded predicted effectors that were highly upregulated during infection. chrY‐frag was not present in wilt‐fragariae; isolates causing this syndrome evolved pathogenicity independently. All origins of F. oxysporum f. sp. fragariae occurred outside of the host’s native range.Our data support the conclusion that HCT is widespread in F. oxysporum, but pathogenicity can also evolve independently. The absence of chrY‐frag in wilt‐fragariae suggests that multiple, distinct pathogenicity chromosomes can confer the same host specificity. The wild progenitors of cultivated strawberry (Fragaria × ananassa) did not co‐evolve with this pathogen, yet we discovered several sources of genetic resistance.

Transient Transgenics: An Efficient Method to Identify Gene Regulatory Elements.

We describe a novel, efficient method to identify cis-acting DNA sequences that drive cell-specific gene expression during development. We utilize transfer of Bacterial Artificial Chromosome (BAC) genomic DNAs, modified to contain a reporter gene, into fertilized mouse embryos and placing the injected embryos into pseudopregnant recipient females. The embryos are allowed to develop in utero for defined times after which they are collected for analysis. Using DNAs containing the LacZ reporter gene facilitates the analysis of gene activity through microscopy of intact embryos and subsequent sectioning of the stained embryos. With this technique cis-element activity can be identified and evaluated through further mutational analysis of the injected BAC DNA. This allows the identification of important gene regulatory domains that specify stage-specific gene expression in the developing embryo.

Identification of Chromosomes and Chromosome Rearrangements in Crop Brassicas and Raphanus sativus: A Cytogenetic Toolkit Using Synthesized Massive Oligonucleotide Libraries.

Crop brassicas include three diploid [Brassica rapa (AA; 2n = 2x = 16), B. nigra (BB; 2n = 2x = 18), and B. oleracea (CC; 2n = 2x = 20)] and three derived allotetraploid species. It is difficult to distinguish Brassica chromosomes as they are small and morphologically similar. We aimed to develop a genome-sequence based cytogenetic toolkit for reproducible identification of Brassica chromosomes and their structural variations. A bioinformatic pipeline was used to extract repeat-free sequences from the whole genome assembly of B. rapa. Identified sequences were subsequently used to develop four c. 47-mer oligonucleotide libraries comprising 27,100, 11,084, 9,291, and 16,312 oligonucleotides. We selected these oligonucleotides after removing repeats from 18 identified sites (500–1,000 kb) with 1,997–5,420 oligonucleotides localized at each site in B. rapa. For one set of probes, a new method for amplification or immortalization of the library is described. oligonucleotide probes produced specific and reproducible in situ hybridization patterns for all chromosomes belonging to A, B, C, and R (Raphanus sativus) genomes. The probes were able to identify structural changes between the genomes, including translocations, fusions, and deletions. Furthermore, the probes were able to identify a structural translocation between a pak choi and turnip cultivar of B. rapa. Overall, the comparative chromosomal mapping helps understand the role of chromosome structural changes during genome evolution and speciation in the family Brassicaceae. The probes can also be used to identify chromosomes in aneuploids such as addition lines used for gene mapping, and to track transfer of chromosomes in hybridization and breeding programs.

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.

Comparative genomics of Fusarium oxysporum f. sp. melonis strains reveals nine lineages and a new sequence type of AvrFom2.

Summary Fusarium oxysporum f. sp. melonis (Fom) is one of the most important pathogens of melon worldwide. In this study, we investigated the genomic diversity of Fom. One of the aims was to find clues for the origin(s) and dispersal of clonal lineages and races of Fom. We therefore included a large number of Fom strains from Iran, where melon has been cultivated for at least 5000 years. In 33 new genome sequences of Fom strains from different geographical regions of Iran and across the world, 40 new candidate effector genes were identified. Presence/absence of candidate effector genes and phylogenetic analyses resolved nine Fom lineages. The presence of a highly similar set of effector genes in some distant lineages is suggestive of horizontal chromosome transfer, a process known to occur in the Fusarium oxysporum species complex. Race 1.2, which breaks both Fom1 and Fom2 resistance genes, occurs in three of the nine lineages, two of which are predominant in Iran. We also identified a new sequence type of the AVRFom2 avirulence gene in one lineage. Expression of this sequence type during melon infection and genetic complementation suggest that this sequence type is not recognized by the Fom2 resistance protein.

Establishment of FXS-A9 panel with a single human X chromosome from fragile X syndrome-associated individual

Fragile X syndrome (FXS) is the most common inheritable form of intellectual disability. FMR1, the gene responsible for FXS, is located on human chromosome Xq27.3 and contains a stretch of CGG trinucleotide repeats in its 5′ untranslated region. FXS is caused by CGG repeats that expand beyond 200, resulting in FMR1 silencing via promoter hypermethylation. The molecular mechanism underlying CGG repeat expansion, a fundamental cause of FXS, remains poorly understood, partly due to a lack of experimental systems. Accumulated evidence indicates that the large chromosomal region flanking a CGG repeat is critical for repeat dynamics. In the present study, we isolated and introduced whole human X chromosomes from healthy, FXS premutation carriers, or FXS patients who carried disease condition-associated CGG repeat lengths, into mouse A9 cells via microcell-mediated chromosome transfer. The CGG repeat length-associated methylation status and human FMR1 expression in these monochromosomal hybrid cells mimicked those in humans. Thus, this set of A9 cells containing CGG repeats from three different origins (FXS-A9 panel) may provide a valuable resource for investigating a series of genetic and epigenetic CGG repeat dynamics during FXS pathogenesis.

Genomic differences between the new Fusarium oxysporum f. sp. apii (Foa) race 4 on celery, the less virulent Foa races 2 and 3, and the avirulent on celery f. sp. coriandrii

BackgroundMembers of the F. oxysporium species complex (FOSC) in the f. sp. apii (Foa) are pathogenic on celery and those in f. sp. coriandrii (Foci) are pathogenic on coriander (=cilantro). Foci was first reported in California in 2005; a new and highly aggressive race 4 of Foa was observed in 2013 in California. Preliminary evidence indicated that Foa can also cause disease on coriander, albeit are less virulent than Foci. Comparative genomics was used to investigate the evolutionary relationships between Foa race 4, Foa race 3, and the Foci, which are all in FOSC Clade 2, and Foa race 2, which is in FOSC Clade 3.ResultsA phylogenetic analysis of 2718 single-copy conserved genes and mitochondrial DNA sequence indicated that Foa races 3 and 4 and the Foci are monophyletic within FOSC Clade 2; these strains also are in a single somatic compatibility group. However, in the accessory genomes, the Foci versus Foa races 3 and 4 differ in multiple contigs. Based on significantly increased expression of Foa race 4 genes in planta vs. in vitro, we identified 23 putative effectors and 13 possible pathogenicity factors. PCR primers for diagnosis of either Foa race 2 or 4 and the Foci were identified. Finally, mixtures of conidia that were pre-stained with different fluorochromes indicated that Foa race 4 formed conidial anastomosis tubes (CATs) with Foci. Foa race 4 and Foa race 2, which are in different somatic compatibility groups, did not form CATs with each other.ConclusionsThere was no evidence that Foa race 2 was involved in the recent evolution of Foa race 4; Foa race 2 and 4 are CAT-incompatible. Although Foa races 3 and 4 and the Foci are closely related, there is no evidence that either Foci contributed to the evolution of Foa race 4, or that Foa race 4 was the recent recipient of a multi-gene chromosomal segment from another strain. However, horizontal chromosome transfer could account for the major difference in the accessory genomes of Foa race 4 and the Foci and for their differences in host range.

Pluripotent stem cell-based gene therapy approach: human de novo synthesized chromosomes.

A novel approach in gene therapy was introduced 20years ago since artificial non-integrative chromosome-based vectors containing gene loci size inserts were engineered. To date, different human artificial chromosomes (HAC) were generated with the use of de novo construction or "top-down" engineering approaches. The HAC-based therapeutic approach includes ex vivo gene transferring and correction of pluripotent stem cells (PSCs) or highly proliferative modified stem cells. The current progress in the technology of induced PSCs, integrating with the HAC technology, resulted in a novel platform of stem cell-based tissue replacement therapy for the treatment of genetic disease. Nowadays, the sophisticated and laborious HAC technology has significantly improved and is now closer to clinical studies. In here, we reviewed the achievements in the technology of de novo synthesized HACs for a chromosome transfer for developing gene therapy tissue replacement models of monogenic human diseases.

Linear, funnel, and multiple funnel schemes for stacking chromosomes that carry targeted recombinations in plants.

Up to five chromosomes that carry targeted recombinations can be stacked via a multiple funnel scheme, provided that the probabilities of inheriting intact chromosomes from donor parents are high. Targeted recombination involves inducing or selecting for recombination events at specific points in the genome to maximize genetic gain. Practical application of targeted recombination requires efficient breeding strategies to stack multiple chromosomes that carry such recombinations. Our objectives were to determine how many chromosomes with targeted recombinations can be feasibly stacked in a breeding program, and how the feasibility of stacking is affected by the crossing design, homozygosity versus heterozygosity of the donor lines, size of the chromosomal segment showing recombination, and probability of an intact chromosome being inherited. Based on a genetic model for maize (Zea mays L.) with 10 pairs of chromosomes, we examined different crossing schemes by simulation experiments and analytical studies with the goal of minimizing the number of generations and population sizes required for stacking. We found that targeted recombinations on up to five chromosomes can be stacked within practical constraints on time and resources. Linear and funnel schemes were less efficient than a multiple funnel scheme, which involved making all possible crosses in the first generation and stacking two additional chromosomes across multiple lines in subsequent generations. Homozygosity versus heterozygosity of the donor lines did not affect stacking efficiency. Population sizes and stacking efficiency were largely determined by the probability of intact chromosomal transfer from a donor parent to offspring. Such probability increased as the size of the chromosome segment from the donor decreased. When the probability of inheriting an intact chromosome was less than 0.15, population sizes needed for stacking became infeasibly large.

A non-mosaic transchromosomic mouse model of down syndrome carrying the long arm of human chromosome 21.

Animal models of Down syndrome (DS), trisomic for human chromosome 21 (HSA21) genes or orthologs, provide insights into better understanding and treatment options. The only existing transchromosomic (Tc) mouse DS model, Tc1, carries a HSA21 with over 50 protein coding genes (PCGs) disrupted. Tc1 is mosaic, compromising interpretation of results. Here, we "clone" the 34 MB long arm of HSA21 (HSA21q) as a mouse artificial chromosome (MAC). Through multiple steps of microcell-mediated chromosome transfer, we created a new Tc DS mouse model, Tc(HSA21q;MAC)1Yakaz ("TcMAC21"). TcMAC21 is not mosaic and contains 93% of HSA21q PCGs that are expressed and regulatable. TcMAC21 recapitulates many DS phenotypes including anomalies in heart, craniofacial skeleton and brain, molecular/cellular pathologies, and impairments in learning, memory and synaptic plasticity. TcMAC21 is the most complete genetic mouse model of DS extant and has potential for supporting a wide range of basic and preclinical research.

Centromere identity and function put to use: construction and transfer of mammalian artificial chromosomes to animal models.

Mammalian artificial chromosomes (MACs) are widely used as gene expression vectors and have various advantages over conventional expression vectors. We review and discuss breakthroughs in MAC construction, initiation of functional centromeres allowing their faithful inheritance, and transfer from cell culture to animal model systems. These advances have contributed to advancements in synthetic biology, biomedical research, and applications in industry and in the clinic.