RESUMEN
A 'genomically' humanized animal stably maintains and functionally expresses the genes on human chromosome fragment (hCF; <24 Mb) loaded onto mouse artificial chromosome (MAC); however, cloning of hCF onto the MAC (hCF-MAC) requires a complex process that involves multiple steps of chromosome engineering through various cells via chromosome transfer and Cre-loxP chromosome translocation. Here, we aimed to develop a strategy to rapidly construct the hCF-MAC by employing three alternative techniques: (i) application of human induced pluripotent stem cells (hiPSCs) as chromosome donors for microcell-mediated chromosome transfer (MMCT), (ii) combination of paclitaxel (PTX) and reversine (Rev) as micronucleation inducers and (iii) CRISPR/Cas9 genome editing for site-specific translocations. We achieved a direct transfer of human chromosome 6 or 21 as a model from hiPSCs as alternative human chromosome donors into CHO cells containing MAC. MMCT was performed with less toxicity through induction of micronucleation by PTX and Rev. Furthermore, chromosome translocation was induced by simultaneous cleavage between human chromosome and MAC by using CRISPR/Cas9, resulting in the generation of hCF-MAC containing CHO clones without Cre-loxP recombination and drug selection. Our strategy facilitates rapid chromosome cloning and also contributes to the functional genomic analyses of human chromosomes.
Asunto(s)
Clonación Molecular , Animales , Cricetinae , Humanos , Ratones , Cromosomas Artificiales , Clonación Molecular/métodos , Cricetulus , Sistemas CRISPR-Cas , ADN , Edición Génica , Células Madre Pluripotentes Inducidas , Translocación GenéticaRESUMEN
Microcell-mediated chromosome transfer is an attractive technique for transferring chromosomes from donor cells to recipient cells and has enabled the generation of cell lines and humanized animal models that contain megabase-sized gene(s). However, improvements in chromosomal transfer efficiency are still needed to accelerate the production of these cells and animals. The chromosomal transfer protocol consists of micronucleation, microcell formation, and fusion of donor cells with recipient cells. We found that the combination of Taxol (paclitaxel) and reversine rather than the conventional reagent colcemid resulted in highly efficient micronucleation and substantially improved chromosomal transfer efficiency from Chinese hamster ovary donor cells to HT1080 and NIH3T3 recipient cells by up to 18.3- and 4.9-fold, respectively. Furthermore, chromosome transfer efficiency to human induced pluripotent stem cells, which rarely occurred with colcemid, was also clearly improved after Taxol and reversine treatment. These results might be related to Taxol increasing the number of spindle poles, leading to multinucleation and delaying mitosis, and reversine inducing mitotic slippage and decreasing the duration of mitosis. Here, we demonstrated that an alternative optimized protocol improved chromosome transfer efficiency into various cell lines. These data advance chromosomal engineering technology and the use of human artificial chromosomes in genetic and regenerative medical research.
RESUMEN
Dystrophin maintains membrane integrity as a sarcolemmal protein. Dystrophin mutations lead to Duchenne muscular dystrophy, an X-linked recessive disorder. Since dystrophin is one of the largest genes consisting of 79 exons in the human genome, delivering a full-length dystrophin using virus vectors is challenging for gene therapy. Human artificial chromosome is a vector that can load megabase-sized genome without any interference from the host chromosome. Chimeric mice carrying a 2.4-Mb human dystrophin gene-loaded human artificial chromosome (DYS-HAC) was previously generated, and dystrophin expression from DYS-HAC was confirmed in skeletal muscles. Here we investigated whether human dystrophin expression from DYS-HAC rescues the muscle phenotypes seen in dystrophin-deficient mice. Human dystrophin was normally expressed in the sarcolemma of skeletal muscle and heart at expected molecular weights, and it ameliorated histological and functional alterations in dystrophin-deficient mice. These results indicate that the 2.4-Mb gene is enough for dystrophin to be correctly transcribed and translated, improving muscular dystrophy. Therefore, this technique using HAC gives insight into developing new treatments and novel humanized Duchenne muscular dystrophy mouse models with human dystrophin gene mutations.
Asunto(s)
Cromosomas Artificiales Humanos , Distrofina , Distrofia Muscular de Duchenne , Animales , Humanos , Ratones , Cromosomas Artificiales Humanos/genética , Modelos Animales de Enfermedad , Distrofina/genética , Distrofina/metabolismo , Ratones Endogámicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Distrofia Muscular de Duchenne/metabolismo , Sarcolema/metabolismoRESUMEN
Mammalian artificial chromosomes have enabled the introduction of extremely large amounts of genetic information into animal cells in an autonomously replicating, nonintegrating format. However, the evaluation of human artificial chromosomes (HACs) as novel tools for curing intractable hereditary disorders has been hindered by the limited efficacy of the delivery system. We generated dystrophin gene knockout (DMD-KO) pigs harboring the HAC bearing the entire human DMD via a somatic cell cloning procedure (DYS-HAC-cloned pig). Restored human dystrophin expression was confirmed by immunofluorescence staining in the skeletal muscle of the DYS-HAC-cloned pigs. Viability at the first month postpartum of the DYS-HAC-cloned pigs, including motor function in the hind leg and serum creatinine kinase level, was improved significantly when compared with that in the original DMD-KO pigs. However, decrease in systemic retention of the DYS-HAC vector and limited production of the DMD protein might have caused severe respiratory impairment with general prostration by 3 months postpartum. The results demonstrate that the use of transchromosomic cloned pigs permitted a straightforward estimation of the efficacy of the DYS-HAC carried in affected tissues/organs in a large-animal disease model, providing novel insights into the therapeutic application of exogenous mammalian artificial chromosomes.
RESUMEN
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 (MMCT) of HACs/MACs has been achieved in various human cells that include human immortalised mesenchymal stem cells (hiMSCs) and human induced pluripotent stem cells (hiPSCs). However, the conventional strategy of gene introduction with HACs/MACs requires 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 MMCT. To overcome these limitations and accelerate chromosome vector-based functional assays 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 these preprepared HAC/MAC-introduced cell lines via the Cre-loxP system or simultaneous insertion of multiple gene-loading vectors. 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 this HAC/MAC-containing cell line panel has dramatically simplified and accelerated gene introduction via HACs/MACs.