Correction of a genetic defect in multipotent germline stem cells using a human artificial chromosome

Abstract

Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance that avoids insertional mutations and the ability to carry large gene inserts including regulatory elements. Multipotent germline stem (mGS) cells have a great potential for gene therapy because they can be generated from an individual's testes, and when reintroduced can contribute to the specialized function of any tissue. As a proof of concept, we herein report the functional restoration of a genetic deficiency in mouse p53−/− mGS cells, using a HAC with a genomic human p53 gene introduced via microcell-mediated chromosome transfer. The p53 phenotypes of gene regulation and radiation sensitivity were complemented by introducing the p53-HAC and the cells differentiated into several different tissue types in vivo and in vitro. Therefore, the combination of using mGS cells with HACs provides a new tool for gene and cell therapies. The next step is to demonstrate functional restoration using animal models for future gene therapy.

Introduction

Homologous recombination has been used for the gene restoration of various genetic defects in embryonic stem (ES) cells,1 but the frequency of homologous recombination in somatic cells is much lower than that in ES cells.2 Furthermore, gene defects with unknown sites of mutation and those involving large deletions cannot be restored by homologous recombination.3, 4, 5 Conventional gene transfer techniques using viral, plasmid, P1 phage-derived artificial chromosome (PAC), bacterial artificial chromosome (BAC) and yeast artificial chromosome (YAC) can also insert DNA randomly into the host genome,6 possibly even causing cancer.7 The use of human artificial chromosomes (HACs) as a vector for gene therapy may solve these problems, because HACs exhibit several important characteristics desired for an ideal gene therapy vector, including stable episomal maintenance and the capacity to carry large genomic loci with regulatory elements, thus allowing the physiological regulation of the introduced gene similar to the native chromosome.8, 9, 10 Although embryonic stem cells for gene therapy may be created for a patient using the nuclear transfer technique, there are many ethical concerns associated with this practice.11, 12 However, multipotent germline stem (mGS) cells successfully generated from the neonatal mouse testis have ES cell-like multipotency.13 If this technology also becomes applicable to the human testis, gene therapy with mGS cells will have advantages over nuclear transfer ES (ntES) cell-mediated gene therapy with respect to ethical problems as well as providing a genetic match with the patient, thus decreasing the likelihood of immune rejection. In this study, we establish a HAC-mediated genomic transfer (HMGT) system as a paradigm for treatment of a genetic disorder by combining mGS cells with a HAC vector containing the normal version of a defective gene.

Results

Construction of HPRT-HAC and the functional analysis

We previously developed a novel HAC vector from normal human chromosome 21 (hChr. 21), using a top-down approach of engineering the chromosome by deleting almost all genes but retaining the centromere and telomeres.9 This HAC vector also contains a cloning site, 3′neo-loxP, into which circular DNA can be reproducibility inserted by using the Cre-loxP system. Fortunately, one of the human BAC/PAC libraries constructed for the human genome sequencing project,14 RPCI-6, has a 5′neo-loxP site, thus allowing the BAC/PAC inserts to transfer readily into the HAC vector (Figure 1). To test whether the RPCI-6 library inserts transferred into our HAC vector are functional, we selected the hypoxanthine phosphoribosyl transferase (HPRT) gene for cloning into the HAC as a model. Out of about 74 000 PAC clones, 7 positive clones were detected by membrane blotting. PCR analyses using 5′-, internal- and 3′-HPRT specific primers showed that 4 out of the 7 clones contained the full-length HPRT gene (Supplementary Table 1). The HPRT PAC clones and a Cre-expression vector were co-transfected into CHO cells containing the HAC vector, and recombinant clones were selected using G418 over a period of 7 days (Figure 2a, Table 1). The drug-resistant clones were positive by PCR with HPRT-specific primers and by fluorescence in situ hybridization (FISH) analysis, and expressed the human HPRT gene by reverse transcription (RT)–PCR analysis (Figures 2b and c). To confirm that the HPRT-HAC was functional, it was introduced into the HPRT-deficient human HeLa cell line,15 D98OR, by microcell-mediated chromosome transfer (MMCT). FISH analysis showed that the HPRT-HAC was successfully transferred into the D98OR cells (Figure 2d). Out of the seven drug-resistant clones, three clones were resistant to HAT but not to 6-thioguanine, thus suggesting that the HPRT-PAC gene in HAC was functionally expressed in the D98OR cells (Supplementary Table 2). The mitotic and expression stability of the HPRT-HAC in D98OR (HPRT-HAC) cells were demonstrated by growing the cells for 4 weeks (approximately 30 population doublings) and then confirming the functional HPRT by HAT-resistance and FISH analysis in 3 independent clones (Figure 2e, Supplementary Figure 1). To investigate the structural integrity of the HPRT-HAC during the transfer from the donor to recipient cells by microcell-fusion and the long-term culture, Southern blot analyses were performed with genomic DNA isolated from the HAC donor CHO cells and human microcell hybrids, digested with BamHI. The restriction fragments were size-separated by pulsed-field gel electrophoresis (PFGE), Southern blotted and hybridized with a probe to α-satellite derived from human chromosome 21.16 In D98OR cells carrying HPRT-HAC, restriction fragments belonging to the HAC were consistent in three independent hybrid clones at an early and late passage (Supplementary Figure 2a). This indicates that HPRT-HAC was correctly transferred from the donor to recipient cells, and that the integrity of HAC was maintained during long-term in vitro serial culture.

Figure 1
figure1

Schematic diagram of the PAC-HAC system for gene delivery. A PAC clone library containing a desired human gene was screened using membrane blotting and PCR. PAC clones containing the desired human gene were cloned into an hChr.21-derived HAC vector by using the Cre-loxP system. Multipotent germline stem (mGS) cells were isolated from mice with genetic defects, and were then genetically restored by transferring the PAC-HAC vector into the cells. The mGS cells containing the PAC-HAC vector were able to differentiate into various cell types in vitro.

Figure 2
figure2

Construction of HPRT-HAC and functional analysis. (a) A schematic diagram of the insertion of RPCI-6 PAC containing the desired gene into the HAC vector. (b) Representative genomic PCR and RT-PCR data for detecting the HPRT-HAC in CHO cells. HT1080 and CHO (HAC) cells were used as positive and negative controls, respectively. β-Actin was used as an internal control. (c and d) Fluorescence in situ hybridization (FISH) analyses for CHO (HPRT-HAC) and D98OR (HPRT-HAC) cells. Digoxigenin-labeled human COT-1 DNA (red) was used to detect HAC in CHO cells (c). Biotin-labeled RP6-114J23 (green) was used to detect the HPRT gene in HACs in CHO (c) and D98OR (d) cells. Chromosomal DNA was counterstained with DAPI. An arrow indicates the HPRT-HAC and the inset shows an enlarged image of the HPRT-HAC. (e) Mitotic stability of the HPRT-HAC in D98OR cells. Mitotic stability was determined by FISH using the biotin-labeled RP6-114J23 after a long period of culture without selection. See online version for color figure.

Table 1 Transfection of the PAC library into CHO cells containing a HAC vector

Stability of HACs in mouse in vivo and in vitro

Next, to demonstrate that HACs can be used for functional analysis in mice, we introduced the parental HAC into mouse ES cells (Supplementary Figure 3a). FISH analysis showed that the HAC was very stable even after long-term culture in vitro (Supplementary Figure 3b). The ES cells were induced to differentiate into neural cells in vitro by using the stromal cell-derived inducing activity (SDIA) method17 (Supplementary Figure 3c). Chimeric mice were produced from the ES cells containing the HAC, and FISH analysis showed that the HAC was present in all the tissues examined (brain, spleen and liver; Supplementary Figure 3d). Therefore, the parental HAC does not interfere with normal development and is stable in mice.

Construction of p53-HAC and the expression analysis

Previously, we first established a novel method to produce mGS cells using p53 deficient mice,13 but mGS cells with other genetic defects have not been established yet. Therefore, we utilized this mouse p53-dificient mGS (p53−/− mGS) cells as a model for functional complementation of genetic defects by the HMGT system. The human p53 gene was cloned into the HAC vector (Table 1 and Supplementary Table 1). PCR, FISH and RT-PCR analyses confirmed that the human p53 gene region was successfully cloned into the HAC and expressed in CHO cells (Figure 3a,b). To determine whether the p53-HAC affects the differentiation in vivo and whether a p53 tissue-specific isoform is expressed, the p53-HAC was transferred into wild-type mouse ES and mGS cells by MMCT (Supplementary Table 2, Figure 3c and, Supplementary Figure 4a). Chimeric mice were produced using ES (p53-HAC) and mGS (p53-HAC), and chimeras with various forms of coat color chimerism were obtained (Supplementary Table 3). FISH and genomic PCR analyses showed that the p53-HAC was retained in all tissues examined, and that about 50% of the cells contained p53-HAC (Figures 3d and e). The incidence was consistent with the coat color chimerism. RT-PCR analyses showed that wild-type human p53 and the splice isoforms were expressed in a tissue-specific manner (Figure 3e), thus suggesting that the PAC-derived p53 gene on the HAC enabled appropriate tissue-specific transcriptional expression of the human p53 gene in mice, despite the human origin of the genomic p53.18, 19

Figure 3
figure3

Construction of p53-HAC and expression analysis in chimeric mice with the HAC. (a) Representative genomic PCR and RT-PCR data for the detection of p53-HAC in CHO cells. HT1080 and CHO (HAC) cells were used as positive and negative controls, respectively. β-Actin was used as an internal control. (bd) Fluorescence in situ hybridization (FISH) analyses for CHO (p53-HAC), ES (p53-HAC) and kidney cells of chimeric mice. Digoxigenin-labeled human COT-1 DNA (red) was used to detect HAC in CHO (b), ES (c), and chimeric kidney cells (d). Biotin-labeled RP6-6J15 (green) was used to detect the p53 gene on the HAC in CHO (b) and ES cells (c). Chromosomal DNA was counterstained with DAPI. The inset shows an enlarged image of the p53-HAC. (e) Representative genomic PCR and RT-PCR data for detection of the p53-HAC in each chimeric tissue. GAPDH was used as an internal control. cDNA and DNA from C57BL/6 mouse tissues were used as negative controls. ES and ES (p53-HAC) cells were used in genomic PCR as negative and positive controls, respectively. See online version for color figure.

Functional analysis in p53−/− mGS cell containing the p53-HAC

The p53-HAC was then introduced into mouse p53−/− mGS cells using MMCT. FISH analyses showed that the p53-HAC was present as an individual chromosome in p53−/− mGS cells (Figure 4a). To confirm whether the transferred p53 trans-activates p53-related genes, both western blot (WB) and real-time PCR analyses were performed for the p53−/− mGS (p53-HAC) cells after X-ray irradiation. The activation of human p53 and mouse p21, p53-related genes, was detected in p53−/− mGS (p53-HAC) cells, but not in p53−/− mGS cells (Figure 4b and Supplementary Figure 4b). To test the function of human p53 in p53−/− mGS cells, we performed a radiosensitivity assay for the p53−/− mGS (p53-HAC) and the p53−/− mGS cells. The p53−/− mGS cells had a significantly higher survival rate after irradiation as compared with two independent p53−/− mGS (p53HAC) cell lines (Figure 4c). These data suggest that the HAC vector containing a p53 gene mediates the production of functional human p53 protein, and that p53-HAC was able to rescue the X-ray-resistant phenotype of the p53−/− mGS cells. To investigate the structural integrity of p53-HAC, Southern blot analyses were performed with genomic DNA isolated from the p53-HAC donor CHO cells and mouse microcell-hybrids, digested with BamHI. The restriction fragments from the p53-HAC were consistent in either mouse ES or p53−/− mGS cells, with an exception in a mGS clone p53−/− mGS (p53-HAC) 1 (Supplementary Figure 2b), in which the functional expression of the introduced p53 was observed. This indicates that inert rearrangements may take place, although the expression of the introduced gene into the loxP site is not affected.

Figure 4
figure4

Transfer of the p53-HAC into p53−/− mGS cells and functional analyses. (a) Fluorescence in situ hybridization (FISH) analysis of the p53−/− mGS (p53-HAC) cells. Digoxigenin-labeled human COT-1 DNA (red) and biotin-labeled RP6-6J15 (green) were used to detect the HAC and the p53 gene on the HAC, respectively, in the p53−/− mGS cells. The inset shows an enlarged image of the p53-HAC. (b) WB analyses of the p53−/− mGS (p53-HAC) cells after X-ray irradiation (8 Gy). The irradiated cells were collected after 0, 2, 4 and 6 h and used for WB analyses. Anti-p53ser15 and anti-p21 antibodies were used to detect human p53 and mouse p21, respectively. Anti-tubulin antibody was used as an internal control. (c) The survival curve of the p53−/− mGS (p53-HAC) and p53−/− mGS cells after X-ray irradiation. Each measurement represents the mean±s.d. of 3 independent experiments. A statistical analysis was performed using a two-tailed Student's t-test. *P<0.05; **P<0.001; (d) Histological analyses of teratomas derived from p53−/− mGS (p53-HAC) cells (hematoxylin and eosin stain). Five weeks after cell transplantation, structures originating from all three germ layers were found in the teratoma. Sq, squamous epithelium (ectodermal derivative); Ca, cartilage (mesodermal derivative); Sk, skeletal muscle (mesodermal derivative); Os, osteoid (mesodermal derivative); Gr, glandular epithelium (endodermal derivative); Ci, ciliated epithelium (endodermal derivative). (e) FISH analysis of teratoma derived from p53−/− mGS (p53-HAC) cells. Digoxigenin-labeled human COT-1 DNA (red) was used to detect the HAC in the tissue. (f) WB analyses of teratoma derived from the p53−/− mGS (p53-HAC) cells. Anti-p53 antibody was used to detect the human p53. Anti-tubulin antibody was used as an internal control. See online version for color figure.

Next, to determine whether the p53−/− mGS (p53-HAC) cells can differentiate in vitro, we used the SDIA methods to induce neuronal differentiation. Immunological analysis revealed that Tuj-1, a neural cell marker, was detected on day 7 as expected (Supplementary Figure 5a). With respect to neural differentiation, the p53−/− mGS (p53-HAC) cells differentiated to a greater extent in vitro compared with the parental p53−/− mGS cells (Supplementary Figure 5b). Furthermore, to determine whether p53−/− mGS (p53-HAC) cells could differentiate into all three embryonic germ layers (endoderm, mesoderm and ectoderm), the cells were subcutaneously injected into nude mice. The transplanted p53−/− mGS (p53-HAC) cells gave rise to typical teratomas (n=5). Histological analyses showed that the tumors contained all three embryonic germ layers, including squamous epithelium, cartilage, skeletal muscle, osteoid, glandular epithelium and ciliated epithelium (Figure 4d), whereas the transplanted parental p53−/− mGS cells were poorly differentiated (data not shown), showing that at least the cell differentiation was restored in vivo by the p53-HAC. FISH analyses showed that the p53-HAC was detected in 90% of cells in tumor tissues (Figure 4e). The expression of the human p53 was detected in the teratomas by WB (Figure 4f) and RT-PCR analyses (data not shown). These data suggest that the presence of p53-HAC did not prevent normal development in vivo or in vitro. These findings are consistent with the results of a previous study by Lin et al.,20 which suggested that p53 induces differentiation of mouse embryonic stem cells by suppressing Nanog expression. Unfortunately, obvious coat color chimeras using the p53−/−(p53-HAC) could not be produced, probably because the parental p53−/− mGS cells had an abnormal karyotype (Supplementary Table 3).

Discussion

HACs have been created by either a top-down approach (engineered chromosome) or a bottom-up approach (de novo artificial chromosome).10 Genomic libraries including PAC, BAC and YAC were loaded to the HACs.21, 22, 23 They were transferred to various types of cells for functional analyses. Prior to the process, however, individual clones from the genomic library were needed to modify for insertion of loxP compatible to the HAC.21, 23 De novo HAC containing desired genomic DNA has been customarily built in HT1080 cell lines,22 from which the delivery of HAC has been hampered to recipient cells. In this study, we directly loaded the ready made genomic library, RPCI-6 PAC with 5′neo-loxP site into HAC, eliminating the recombination step in the yeast or bacteria.

During the investigation of the HAC integrity by Southern blotting with a human chromosome 21-derived alphoid satellite probe, we found a rearrangement of the restriction fragments of the HAC centromere in one of the p53−/− mGS (p53-HAC) clones. This structural change affected neither the expression of the introduced p53 gene nor the stability of the HAC. For the future application of the HAC to the gene therapy protocol, however, any rearrangements that may lead to safety concerns are not desirable even if they are inert. From this study, we now obtained one of the practical criteria to exclude the clones containing a possible structural rearrangement.

A current limitation of this study is that the existing PAC libraries were randomly created and the inserts may contain genes or sequences other than those desired. Selective cloning using the methods of transformation-associated recombination (TAR) cloning or BAC recombineering can be used to overcome this problem.24, 25 In the case of the transfer of megabase-sized genomic DNA such as HLA cluster (3 Mb), DMD gene (2.5 Mb) and p450 gene cluster (1 Mb), we can apply a Cre-loxP mediated chromosomal translocation system.8 Therefore, every human genomic DNA could be cloned by a combination of these cloning systems on HAC and could be transferred into desired cell types using HAC. HAC with the desired human gene can also be transferred to mouse ES cells, and the chimeric mice or offspring will be useful either for the functional analysis in vivo or for a human disease model.26

Although the correction of genetic defect using ntES cells was reported, at least in mice,1 the production of patient-specific ntES cells would be more difficult in the human, and create ethical problems.11 Therefore, the creation of a patient-specific multipotent stem cell without the usage of ntES technology would be important for stem cell-based gene therapy in genetic disorders. In this study, we used mGS cells from neonatal mouse testis as our recipient stem cells. Recently, Guan et al.27 reported isolation of ES-like cells from adult mouse testis. Even more interestingly, ES-like cells can be induced to form from adult mouse fibroblasts by the transfer of four factors.28 In the future, using such individual ES-like stem cells is expected to allow us to overcome the ethical problems that are encountered when using this method for humans.

In conclusion, we demonstrated that the HMGT system is a potential new tool for gene delivery, and that our engineered HAC vector can be transferred by using an MMCT approach to mGS cells to correct a genetic defect in these cells. Advances in efficient methods for differentiation and purification of stem cells, including ES and mGS cells, are anticipated in the future. Using stem cells derived from multiple potential sources combined with the HAC-mediated gene delivery may comprise this useful treatment for genetic defects in a near future.

Materials and methods

Construction of the PAC-HAC vector

The RPCI-6 human PAC library, which was obtained from the BACPAC Resources Center at the Children's Hospital Oakland Research Institute, was screened using either p53cDNA or a PCR derived HPRT-specific probe as described at http://bacpac.chori.org/. Positive PAC clones and Cre-recombinase expression vectors (Invitrogen, Carlsbad, CA, USA) were co-transfected into CHO cells containing a HAC vector using Lipofectamine 2000 (Invitrogen) in accordance with the manufacturer's protocol. The cell culture and colony expansion were performed as described previously.9 The site-specific chromosomal insertion into the HAC was confirmed by PCR and FISH analyses.

Genomic PCR analyses

Genomic DNA was extracted from host cells containing the PAC-HAC using a genomic extraction kit (Gentra Systems, Minneapolis, MN, USA), and PCR was performed using primers as follows: for 5′-HPRT (243 bp), 5′-IndexTermAAAATGGAAGCCACAGGTAGTGCAAGGT-3′ and 5′-IndexTermAGGCTCACTAGGTAGCCGTGGGAATTTT-3′; for internal-HPRT (575 bp), 5′-IndexTermTGCTGGGATTACACGTGTGAACC-3′ and 5′-IndexTermGACTCTGGCTAGAGTTCCTCCTCCC-3′; for 3′-HPRT (219 bp), 5′-IndexTermCATGTCTTATCAGAACCAGGGAGGCAGA-3′ and 5′-IndexTermCAGCTCTGGCCATGAATGTCTTCACATA-3′; for CMV-Neo (318 bp), 5′-IndexTermCGTAACAACTCCGCCCCATT-3′ and 5′-IndexTermGCAGCCGATTGTCTGTTGTG-3′; for 5′-p53 (280 bp), 5′-IndexTermCGAGCTCTTACTTGCTACCCAGCACTGA-3′ and 5′-IndexTermTTGCTCTCAGCTGGATCCTTTCTTCTCA-3′; for internal-p53 (1069 bp), 5′-IndexTermTCCCCTGCCCTCAACAAGATG-3′ and 5′- IndexTermATCACACTGGAAGACTCCAG-3′; for 3′-p53 (205 bp), 5′-IndexTermGGCTCCATTCATAACTCAGGAACCAACC-3′ and 5′-IndexTermGTATCCTGCCACTTCTGATGGACGAAGA-3′.

FISH analyses

FISH analyses were performed using either fixed metaphase or interphase spreads of each cell hybrid using digoxigenin-labeled (Roche, Basel, Switzerland) human COT-1 DNA (Invitrogen) and biotin-labeled PAC DNA, essentially as described previously.19 Chromosomal DNA was counterstained with DAPI (Sigma, St Louis, MD, USA). The images were captured using the Argus system (Hamamatsu Photonics, Hamamatsu, Japan).

RT-PCR analyses

Total RNA was prepared from cultured cells and chimeric tissue specimens using RNeasy columns, according to the manufacturer's instructions (Qiagen, Hilden, Germany), and then was treated with RNase-free DNase I (Wako, Osaka, Japan). First-strand cDNA synthesis was carried out using an oligo-(dT)15 primer and M-MLV reverse transcriptase (Invitrogen). PCR was performed with cDNA using AmpliTaq Gold (Perkin Elmer, Waltham, MA, USA). Amplifications were performed with an annealing temperature of 58 °C for 30 cycles, and then the amplified fragments were resolved on a 2% agarose gel, followed by staining with ethidium bromide. The primer sequences were as follows: for HPRT (200 bp), 5′-IndexTermTCCTCCTCCTGAGCAGTCA-3′ and 5′- IndexTermCATCTCGAGCAAGACGTTCA -3′; for β -actin (165 bp), 5′-IndexTermTGTTACCAACTGGGACGACA-3′ and 5′-IndexTermGGGGTGTTGAAGGTCTCAAA-3′; for GAPDH (722 bp), 5′-IndexTermCCATCTTCCAGGAGCGAGA-3′ and 5′-IndexTermTGTCATACCAGGAAATGAGC-3′; for p53-3′UTR (154 bp), 5′-IndexTermCTCTCCCTCCCCTGCCATTT-3′ and 5′-IndexTermCCATCCTCCTCCCCACAACA-3′; for p53-1 (434 bp), 5′-IndexTermGTGGAAGGAAATTTGCGTG-3′ and 5′- IndexTermCTTTTTGAAAGCTGGTCTGG -3′; for p53-2 (476 bp), 5′-IndexTermGTGGAAGGAAATTTGCGTG-3′ and 5′-IndexTermAGCCTGGGCATCCTTGAGT-3′. Real-time RT-PCR was performed on an ABI 7000 machine using the SyBr Green PCR Master Mix (ABI, Forster City, CA, USA). The PCR conditions consisted of a 10-min hot start at 95 °C, followed by 40 cycles of 30 s at 95 °C and 1 min at 61 °C. The average threshold cycle was determined on the basis of triplicate reactions and the levels of gene expression relative to β-actin were determined as described.29 The primer sequences for p21 were as described previously.30

WB analyses

Protein extracted from cultured cells and tissues was separated by SDS-PAGE on a 10% polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% dry milk and probed with a polyclonal antibody against phospho-p53 (Ser15) (no. 9248; Cell Signaling Technology, Danvers, MA, USA) or a monoclonal antibody against p53 (D07; Novocastra Laboratories, Newcastle, UK). The membrane was then incubated with a horseradish-peroxidase-conjugated secondary antibody and developed with enhanced chemiluminescence PLUS reagent (Amersham, Sunnyvale, CA, USA). To confirm that the amount of protein in each lane was comparable, the membrane was stripped and probed with a monoclonal antibody against α-tubulin (DM-1A; ICN Biomedicals, Costa Mesa, CA, USA).

Radiation survival assay

The cells were irradiated with 0-6 Gy of X-ray using a Hitachi Medico MBR-1505R2 X-ray irradiation machine (Hitachi, Tokyo, Japan). The cells were irradiated under the following conditions: 150 kV, 5 mA, 0.9 Gy/min. The survival curves for each cell line were obtained by measuring the colony-forming abilities of irradiated cell populations as previously described.31 Three independent experiments were performed.

Generation of chimeric mice containing the p53-HAC

p53−/− mGS cells were generated from the testis tissue specimens from p53-deficient neonatal mice.13 MMCT, ES/mGS cell culture and chimera production were performed as described previously.19 Briefly, mouse ES (E14), mGS and p53−/− mGS cells were fused with microcells prepared from donor hybrid Chinese hamster ovary (CHO) cells containing the p53-HAC and selected with Blasticidin S (10 μg ml−1). The transferred p53-HAC in each line was characterized by PCR and FISH analyses. Chimeric mice were produced from the 2 ES cell lines containing the p53-HAC. Five chimeric mice showing 50% coat color chimerism were used for the analyses of expressions in various tissues. All chimeric mice used for expression analyses were 12–18 weeks old. All animal experiments were approved by the Institutional Animal Care and Use Committee of Tottori University.

PFGE and Southern blotting

A high molecular weight DNA for long-range mapping was prepared in agarose gel plugs as described in Trowell et al.32 The plugs were sliced into segments, each containing 0.4 × 106 cells. Agarose-DNA segments were then digested for 18 h with BamHI (Takara; 100 U per segment). The DNA fragments were size separated on a CHEF DR-II apparatus (Bio-Rad Laboratories, Hercules, CA, USA). The run time was 24 h at 6 V cm−1 with a 60–120 s switch time ramp. Human chromosome 21-derived α-satellite clone p11-416 was labeled with α-32P-dCTP by the random priming method (Amersham). Hybridization was carried out at 65 °C in 0.5 M phosphate buffer (0.5 M Na2PO4, 7% SDS, 1 mM EDTA, pH 7.2). Following hybridization, the filters were washed in 40 mM phosphate buffer/1% SDS at 65 °C. The autoradiographs were obtained by an imaging system BAS2500 (Fuji Film, Tokyo, Japan).

Teratoma formation and histology

For subcutaneous injections to produce teratomas, 2 × 106 p53−/− mGS (p53HAC) and p53−/− mGS cells were injected into CD-1 (ICR)-nu mice (Charles River, Yokohama, Japan). After 5–6 weeks, the teratomas were fixed in 20% formalin and processed for paraffin sectioning.

Induction of mGS cell differentiation to neuronal cells and immunocytochemical analysis

The differentiation of ES, ES (HAC), p53−/− mGS and p53−/− mGS (p53-HAC) cells was induced using the SDIA method as described by Kawasaki et al.17 The cells differentiated in chamber slides (Nunc, Roskilde, Denmark) for 7 days were fixed in 4% paraformaldehyde, permeabilized in 0.2% Triton-X100 (Sigma), and treated in Block-ace solution (Snow Brand Milk Products, Tokyo, Japan). Anti-Tuj-1 antibody (Babco, Richmond, CA, USA) was used for the immunocytochemical analysis. Cy-3-conjugated secondary antibodies (Chemicon, Temecula, CA, USA) were used. Three independent experiments were performed and 100 colonies were counted in each experiment to determine the Tuj-1-positive proportion of cells.

Statistical analysis

The data are presented as the mean±s.d. All analyses were performed using the two-tailed Student's t-test. P-values less than 0.05 were considered to be significant.

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Acknowledgements

We thank K Sato for providing the human p53 cDNA; K Hanaoka, S Tsuji, C Okita, H Yamada, Y Iida and J Nakamura for technical assistance and K Tomizuka, A Kurimasa H Kugoh, T Inoue and M Hiratsuka for valuable discussions. This study was supported in part by a Health and Labor Science Research Grant for Research on the Human Genome, Tissue Engineering from the Ministry of Health, Labour and Welfare, Japan (MO), and the 21st Century COE program from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MO).

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Correspondence to M Oshimura.

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Supplementary Information accompanies the paper on Gene Therapy website (http://www.nature.com/gt)

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Keywords

  • human artificial chromosome
  • multipotent germline stem cell
  • microcell-mediated chromosome transfer

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