Highly stable maintenance of a mouse artificial chromosome in human cells and mice

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Highlights

  • A MAC was stably maintained in human HT1080 cells during long term-culture.

  • The MAC vector was stably maintained at least to the F8 generation in mice.

  • The MAC was stably maintained in various tissues derived from old mice.

  • Multiple copies of the MAC were stably retained in mice.

  • The gene expression on the MAC was dependent on the chromosomal copy number.

Abstract

Human artificial chromosomes (HACs) and mouse artificial chromosomes (MACs) display several advantages as gene delivery vectors, such as stable episomal maintenance that avoids insertional mutations and the ability to carry large gene inserts including the regulatory elements. Previously, we showed that a MAC vector developed from a natural mouse chromosome by chromosome engineering was more stably maintained in adult tissues and hematopoietic cells in mice than HAC vectors. In this study, to expand the utility for a gene delivery vector in human cells and mice, we investigated the long-term stability of the MACs in cultured human cells and transchromosomic mice. We also investigated the chromosomal copy number-dependent expression of genes on the MACs in mice. The MAC was stably maintained in human HT1080 cells in vitro during long-term culture. The MAC was stably maintained at least to the F8 and F4 generations in ICR and C57BL/6 backgrounds, respectively. The MAC was also stably maintained in hematopoietic cells and tissues derived from old mice. Transchromosomic mice containing two or four copies of the MAC were generated by breeding. The DNA contents were comparable to the copy number of the MACs in each tissue examined, and the expression of the EGFP gene on the MAC was dependent on the chromosomal copy number. Therefore, the MAC vector may be useful not only for gene delivery in mammalian cells but also for animal transgenesis.

Introduction

The introduction of large genes or gene clusters into mice allows the correct expression of transgenes by including essential remote regulatory elements [1]. Human chromosome fragments (hCFs) derived from normal fibroblasts were used as a vector for animal transgenesis, including the introduction of Mb-sized large genomic inserts into mice via microcell-mediated chromosome transfer (MMCT) technology [2], [3]. Double transchromosomic (Tc) mice containing two individual hCFs carrying IgH and Igk produced antigen-specific human antibodies [4]. However, the mitotic stability of hCFs in mice varies, and large hCFs cannot be transmitted through the germline [2], [5], [6]. Cloning the desired genomic region into the mitotically stable hCF allowed us to generate Tc mice containing multiple large genomic inserts, which could not be cloned using conventional vectors such as plasmids and bacterial artificial chromosomes (BACs) [5], [7]. However, hCFs contain several structurally undefined regions with many endogenous genes, which cause partial trisomy in cells propagating these hCFs. This may affect the physiological gene expression and the normal development. To overcome this, several groups engineered human artificial chromosomes (HACs) by random segmentation or targeted telomere-associated chromosomal fragmentation in homologous recombination-proficient chicken DT40 cells [5], [8], [9], [10]. However, germline transmittable Tc mice containing multiple copies of HACs with Mb-sized large inserts have never been generated, possibly because of the instability of HACs in germ cells. Although hCFs and HACs containing large regions of genomic DNA can be autonomously maintained in Tc mice, their retention rate varies [3], [6], [11], [12], [13], [14]. Thus, we constructed novel mouse artificial chromosome (MAC) vectors from a native mouse chromosome by chromosome engineering to improve the retention rate [15]. Previously, a MAC vector containing the EGFP gene, which can be used to monitor the cells, was used to determine its stability in vivo. The stability of this MAC in mouse tissues and hematopoietic cells was higher than that of other reported mammalian artificial chromosomes including hCFs, HACs, and murine satellite DNA-based artificial chromosomes (mSATACs) [16]. The stability and germline transmission (GT) efficiency of hCFs, HACs, and mSATACs differed with genetic background, generation, age, and sex [4], [6], [12], [14], [17], [18]. MACs will be a powerful tool to generate Tc mice carrying multiple Mb-sized genes for humanized animal models if they have high stability and GT efficiency and if multiple copies can be introduced into mice. Furthermore, if the MAC is stable in human cells, the same MAC containing a desired gene may be used for functional analysis in both mice and human cells. Therefore, we investigated: (i) MAC stability in HT1080 cells, (ii) MAC stability and GT ratio in different genetic backgrounds and sexes, (iii) MAC stability in aged mice, (iv) MAC copy number per cell in tissues, and (v) MAC copy number-dependent gene expression in tissues.

Section snippets

Cell culture

HT1080 were grown in Dulbecco’s modified Eagle’s medium (Sigma, St. Louis, MO, USA) plus 10% fetal bovine serum (FBS). Chinese hamster ovary (CHO) cells containing MAC1 were constructed as previously described [15]. The CHO (MAC1) cells were maintained in Ham’s F-12 nutrient mixture (Invitrogen, Carlsbad, CA, USA) plus 10% FBS with 800 μg/mL G418 (Promega, Tokyo, Japan).

MMCT

MMCT was performed as described previously [2], [19]. CHO cells containing MAC1 were used as donor microcell hybrids. The

Stability of MAC1 in HT1080 cells

We used the MAC1 vector with EGFP to monitor the gene expression and stability of the MAC. To investigate the stability of a MAC in human cells, MAC1 was transferred to the human fibrosarcoma cell line HT1080 by MMCT. Three GFP-positive clones were selected and examined. FISH analyses showed that MAC1 was present as an individual chromosome in the HT1080 cells (Fig. 1A). After the HT1080 (MAC1) cells were cultured for about 3 months with or without selection, the stability of the MAC1 and GFP

Acknowledgements

We wish to thank Kaori Adachi, Yumiko Kumura, Kayo Fujimoto, Kazuki Tanaka, Naoyo Kajitani, Toko Yoshino, Hiromichi Kono, Yukako Sumida, Manami Iitsuka, Madoka Fukuura, Noriyasu Oko, and Ayako Takami for technical assistance, and Yuji Nakayama and Eiji Nanba for critical discussions. This study was supported in part by the Funding Program for Next Generation World-Leading Researchers (NEXT Program) from the Japan Society for the Promotion of Science (JSPS) (Y.K.), JST, CREST (M.O.), and

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