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.


Introduction
Human artificial chromosomes (HACs) and mouse artificial chromosome (MACs) have unique characteristics as vectors for gene delivery, which include stably and independently maintenance without disruption of the host genome and the capacity to carry numerous genes and megabase-sized genomic loci with physiological regulatory elements 1-4 . HAC/MAC technologies have been used for gene and cell therapies of Duchene muscular dystrophy 5-9 and to generate trans-chromosomic (Tc) animals including a mouse model of Down syndrome 10, 11 and humanised drug metabolism [12][13][14][15][16][17] .
Furthermore, several types of HACs have been used in cancer research and drug screening for cancer therapy 18,19 , centromere and telomere function elucidation 2020 , a system of quantitatively tracking epigenetic memory in the field of synthetic biology 21 , and protein production 22 . To accelerate the geneloading of multiple genes into HACs/MACs, we developed several systems for multiple gene insertions, such as simultaneous or sequential integration of multiple gene-loading vectors (SIM system) 23,24 , a multi-integrase (MI) system [25][26][27][28] , and homologous recombination with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) 29 . HACs/MACs are transferrable into desired cells by microcellmediated chromosome transfer (MMCT) 30 . Although MMCT traditionally employs polyethylene glycol 31 , we developed a novel microcell membrane fusion method with the envelope proteins of measles virus (MV) 32,33 , amphotropic virus, and ecotropic virus 34 , which improved the transfer efficiency (1×10 -4 to 1×10 -5 ). However, specialised equipment and a laborious and time-consuming process are required for MMCT of HACs/MACs, because in accordance with each experimental purpose, HACs/MACs with desired genes are constructed in CHO and A9 cells and individually transferred to a target cell line via MMCT, then isolated the clones containing the desired HACs/MACs (Fig. 1a). Therefore, we have previously employed mouse embryonic stem cells that contain a MAC with the MI system to facilitate the generation of Tc mice 35 . Under such circumstances, ready-made human cell lines containing HACs/MACs will be useful platform for simple and stable gene expression. The detailed structures of each HAC/MAC are shown in Supplementary Figure S1. Here, we report the generation of a human cell line panel to facilitate functional analyses of genes of interest using HACs/MACs (Fig. 1b). As representative human cell lines, we used HEK293 (ATCC ® CRL-1573™), which is an immortalised human cell line, and HT1080, which is a cancer cell line, as well as a human immortalised mesenchymal stem cell (hiMSC) line 36,37 and human induced pluripotent stem cell (hiPSC; 201B7) line 38 . We further attempted to adapt the Cre-loxP system (Supplementary MACs, were used in this study ( Supplementary Fig. S1). HACs were derived from human chromosome 21, such as 21HAC1 without EGFP and 21HAC2 with EGFP 39 , and MACs were derived from mouse chromosome 11, such as MAC2 without EGFP, MAC4 with EGFP 35,40,41 , and MAC6 with EGFP. These HACs/MACs had a loxP site and partial HPRT gene as an acceptor site for the SIM system, which enable simultaneous insertion of three circular plasmids. However, conventional gene introduction via HACs/MACs requires transfer of the HACs/MACs with desired gene(s) by MMCT into target cells and there is a major technical difficulty. In this study, we established a human somatic/stem cell line panel (HEK293, HT1080, hiMSCs, and hiPSCs) that contained HACs/MACs ( Fig. 1) (Table 1).  S3c and S3d). These results showed that multiple gene loading into HAC/MAC via SIM system was successfully achieved in our established human cell line panel (Fig. 2a-2f), enabling seamless application of our HAC/MAC technology for gene functional assay in human cells in future.
Characterisation of hiMSCs that contain HACs/MACs and demonstration of gene loading by the Cre-loxP system As hiMSCs with a transferred 21HAC2 (Fig. 2g), which were clones A03 and D11, stably expressed EGFP (Fig. 4a). Various MSC markers were also analysed by RT-PCR analysis in these clones (Fig. 4b). To evaluate the HAC retention ratio after long-term cell culture with or without an antibiotic (blasticidin; Bsd), FISH analysis was performed and results showed stable maintenance of the HAC at population doubling level (PDLs) of 24 and 39, even without Bsd (Fig. 4c). These results indicated that the two hiMSC clones that contained 21HAC2 (hiMSC/21HAC2 A03 and D11) could be used for a platform of gene loading 36 . We validated whether the 21HAC2 could function as a safe harbour for gene expression in these established clones. As an example of functional analysis, we attempted to evaluate the expression level of the transgene promoted by three types of constitutive promoters (PGK, EF1α, and CAG) in 21HAC2 in hiMSCs with defined (single in this study) copy number. Among the drug-resistant clones obtained by transfection with a plasmid that carried each promoter, 10 clones were picked up in order of the fluorescence intensity of mCherry under a fluorescence microscope and used for subsequent analysis to measure each promoter activity. Fluorescence imaging of mCherry-expressing cells with each promoter indicated that CAG and EF1α promoter activities were higher than the PGK promoter activity (Fig. 4d). qRT-PCR of the mRNA expression level of mCherry also indicated that CAG and EF1 promoter activities were higher at 22.4-fold (CAG) and 40.5-fold (EF1α) compared with the PGK promoter activity (n=10) (P < 0.01) (Fig. 4e). There was no significant difference between the activities of CAG and EF1α promoters. These results supported a previous study that compared promoter activity in MSCs with a viral vector system for gene expression 43 . Because the Cre-loxP system had the same adaptor as the SIM system in 21HAC2, the SIM system would be applicable to hiMSCs/21HAC2. These results showed that hiMSCs that contained 21HAC2 were applicable to gene loading and gene functional analyses.
Characterisation of hiPSCs that contained HACs/MACs and demonstration of gene loading by the SIM system hiPSCs (201B7) that contained MAC6-ΔNeoR expressed EGFP (Fig. 5a). We and micronuclei were induced with 0.1 µg/mL colcemid. The detailed MMCT protocol has been described previously 24 . The collected microcells were cocultured and fused with 2 × 10 6 cells of each recipient cell line for 24 hours in a 6-cm dish (Corning, Corning, NY, USA). Then, the fused recipient cells were subcultured into three 10-cm dishes. Drug selection was started with optimal selectable antibiotics after a further 24 hours of incubation. After 14-21 days, drug-resistant colonies were picked up and expanded for the following analyses.

Plasmid construction
To construct pBG-V0b1-ins-ELuc-ins, an EcoRV-digested fragment, which includes an ELuc expression unit from CAG-ELuc, was ligated into pBG-V0b1 linearised with EcoRV. To construct pBG2-V1a-ins-tdtmt-ins and pBG2-V2ains-BFP-ins, each fragment of pCMV-tdTomato (Takara Bio) and pTagBFP2-N (Evrogen) was amplified by PCR with the following primers, F: 5'-    The probes were labelled with digoxigenin (Roche, Basel, Schweiz) and the inserted plasmid vector targeted to the chromosome fragment was labelled with biotin (Roche). The DNA probes were labelled with a nick translation kit (Roche) following the manufacturer's instructions. The detailed protocol has been described previously 24 .

Teratoma formation and histological analysis
The mice were maintained under specific pathogen-free conditions with a 12h light-dark cycle. Human iPSCs (1 × 10 6 ) were subcutaneously transplanted to a testis of anaesthetized severe combined immunodeficiency (SCID) mice (Charles River, Yokohama, Japan). A mixed anaesthetic agent prepared with 0.3 mg/kg of medetomidine hydrochloride, 4 mg/kg of midazolam, and 5 mg/kg of butorphanol tartrate was administered intraperitoneally for the mice.
Teratomas appeared after ~8 weeks. The anaesthetized mice were sacrificed and the teratoma was explanted. Then, the teratoma was fixed with 20% neutral formalin/PBS and processed for paraffin sectioning. The sections were stained with haematoxylin and eosin.

Flowcytometry (FCM analysis)
To evaluate the ratio of cells expressing fluorescent proteins, the cells were analysed by a FCM using Fortessa (BD) LSR X-20 flowcytometer (Beckton Dickinson) equipped with 488nm, 405nm, and 561 nm laser for detection of EGFP, BFP2, and tdTomato, respectively. The ratio of fluorescence-positive cell of each fluorescence protein was determined.

Luciferase assay
The assay was performed with 6 × 10 4 cells in each well of a 96-well plate.

Luciferase activity was measured with Emerald Luc Luciferase Assay
Reagent Neo (TOYOBO) following the manufacturer's instructions.

RT-PCR
Total RNA was isolated with a Nucleospin RNA plus kit (Takara Bio), following the manufacturer's instructions. cDNA was synthesised with a         hiMSCs that contained 21HAC2 were used to attempt insertion of three types of plasmid vectors that contained a red uorescent protein (mCherry) driven by a different promoter with the Cre-loxP system.