Human Cell Line Panel With Human/Mouse Artificial Chromosomes for Functional Analyses of Desired Genes

Human artificial chromosomes (HACs) and mouse artificial chromosomes (MACs) are non-integrating chromosomal gene delivery vectors for molecular biology research. Recently, microcell-mediated chromosome transfer of HACs/MACs has been achieved into various human cells including human immortalised mesenchymal stem cells (hiMSCs) and human induced pluripotent stem cells (hiPSCs). However, the conventional strategy of gene-introduction with HAC/MAC required laborious and time-consuming stepwise isolation of clones for gene loading into HACs/MACs in donor cell lines (CHO and A9) and then transferring the HAC/MAC into cells via microcell-mediated chromosome transfer (MMCT). To overcome these limitations and accelerate chromosome vector based functional assay in human cells, we established various human cell lines (HEK293, HT1080, hiMSCs, and hiPSCs) with HACs/MACs that harbour a gene-loading site via MMCT. Model genes, such as tdTomato, TagBFP2, and ELuc, were introduced into the premade HAC/MAC-introduced cell lines via the Cre-loxP system or simultaneous insertion of multiple gene-loading vectors (SIM system). The model genes on the HACs/MACs were stably expressed and the HACs/MACs were stably maintained in the cell lines. Thus, our strategy using the HAC/MAC-containing cell line panel has dramatically simplified and accelerated gene introduction via HACs/MACs, thereby facilitating functional analyses of introduced genes.

Humanized UGT2 and CYP3A transchromosomic rats for improved prediction of human drug metabolism

Although “genomically” humanized animals are invaluable tools for generating human disease models as well as for biomedical research, their development has been mainly restricted to mice via established transgenic-based and embryonic stem cell-based technologies. Since rats are widely used for studying human disease and for drug efficacy and toxicity testing, humanized rat models would be preferred over mice for several applications. However, the development of sophisticated humanized rat models has been hampered by the difficulty of complex genetic manipulations in rats. Additionally, several genes and gene clusters, which are megabase range in size, were difficult to introduce into rats with conventional technologies. As a proof of concept, we herein report the generation of genomically humanized rats expressing key human drug-metabolizing enzymes in the absence of their orthologous rat counterparts via the combination of chromosome transfer using mouse artificial chromosome (MAC) and genome editing technologies. About 1.5 Mb and 700 kb of the entire UDP glucuronosyltransferase family 2 and cytochrome P450 family 3 subfamily A genomic regions, respectively, were successfully introduced via the MACs into rats. The transchromosomic rats were combined with rats carrying deletions of the endogenous orthologous genes, achieved by genome editing. In the “transchromosomic humanized” rat strains, the gene expression, pharmacokinetics, and metabolism observed in humans were well reproduced. Thus, the combination of chromosome transfer and genome editing technologies can be used to generate fully humanized rats for improved prediction of the pharmacokinetics and drug–drug interactions in humans, and for basic research, drug discovery, and development.

The multi-speed genome ofFusarium oxysporumreveals association of histone modifications with sequence divergence and footprints of past horizontal chromosome transfer events

Fusarium oxysporumis an economically important pathogen causing wilting or rotting disease symptoms in a large number of crops. It is proposed to have a structured, “two-speed” genome: i.e. regions containing genes involved in pathogenicity cluster with transposons on separate accessory chromosomes. This is hypothesized to enhance evolvability. Given the continuum of adaptation of all the genes encoded in a genome, however, one would expect a more complex genome structure. By comparing the genome of reference strain Fol4287 to those of 58 otherFusarium oxysporumstrains, we found that some Fol4287 accessory chromosomes are lineage-specific, while others occur in multiple lineages with very high sequence similarity - but only in strains that infect the same host as Fol4287. This indicates that horizontal chromosome transfer has been instrumental in past host-switches. Unexpectedly, we found that the sequence of the three smallest core chromosomes (Chr. 11, 12 and 13) is more divergent than that of the other core chromosomes. Moreover, these chromosomes are enriched in genes involved in metabolism and transport and genes that are differentially regulated during infection. Interestingly, these chromosomes are –like the accessory chromosomes– marked by histone H3 lysine 27 trimethylation (H3K27me3) and depleted in histone H3 lysine 4 dimethylation (H3K4me2). Detailed genomic analyses revealed a complex, “multi-speed genome” structure inFusarium oxysporum. We found a strong association of H3K27me3 with elevated levels of sequence divergence that is independent of the presence of repetitive elements. This provides new leads into how clustering of genes evolving at similar rates could increase evolvability.Author summaryFungi that cause disease on plants are an increasingly important threat to food security. New fungal diseases emerge regularly. The agricultural industry makes large investments to breed crops that are resistant to fungal infections, yet rapid adaptation enables fungal pathogens to overcome this resistance within a few years or decades. It has been proposed that genome ‘compartmentalization’ of plant pathogenic fungi, in which infection-related genes are clustered with transposable elements (or ‘jumping genes’) into separate, fast-evolving regions, enhances their adaptivity. Here, we aimed to shed light on the possible interplay between genome organization and adaptation. We measured differences in sequence divergence and dispensability between and within individual chromosomes of the important plant pathogenFusarium oxysporum. Based on these differences we defined four distinct chromosomal categories. We then mapped histone modifications and gene expression levels under different conditions for these four categories. We found a ‘division of labor’ between chromosomes, where some are ‘pathogenicity chromosomes’ - specialized towards infection of a specific host, while others are enriched in genes involved in more generic infection-related processes. Moreover, we confirmed that horizontal transfer of pathogenicity chromosomes likely plays an important role in gain of pathogenicity. Finally, we found that a specific histone modification is associated with increased sequence divergence.