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• W2035554773 abstract "Human neocentromeres are functional centromeres that are devoid of the typical human centromeric α-satellite DNA. We have transferred a 60-Mb chromosome 10-derived neocentric marker chromosome, mardel(10), and its truncated 3.5-Mb derivative, NC-MiC1, into mouse embryonic stem cell and have demonstrated a relatively high structural and mitotic stability of the transchromosomes in a heterologous genetic background. We have also produced chimeric mice carrying mardel(10) or NC-MiC1. Both transchromosomes were detected as intact episomal entities in a variety of adult chimeric mouse tissues including hemopoietic stem cells. Genes residing on these transchromosomes were expressed in the different tissues tested. Meiotic transmission of both transchromosomes in the chimeric mice was evident from the detection of DNA from these chromosomes in sperm samples. In particular, germ line transmission of NC-MiC1 was demonstrated in the F1 embryos of the chimeric mice. Variable (low in mardel(10)- or NC-MiC1-containing embryonic stem cells and chimeric mouse tissues and relatively high in NC-MiC1-containing F1 embryos) levels of missegregation of these transchromosomes were detected, suggesting that they are not optimally predisposed to full mitotic regulation in the mouse background, particularly during early embryogenesis. These results provide promising data in support of the potential use of neocentromere-based human marker chromosomes and minichromosomes as a tool for the study of centromere, neocentromere, and chromosome biology and for gene therapy studies in a mouse model system. They also highlight the need to further understand and overcome the factors that are responsible for the definable rates of instability of these transchromosomes in a mouse model. Human neocentromeres are functional centromeres that are devoid of the typical human centromeric α-satellite DNA. We have transferred a 60-Mb chromosome 10-derived neocentric marker chromosome, mardel(10), and its truncated 3.5-Mb derivative, NC-MiC1, into mouse embryonic stem cell and have demonstrated a relatively high structural and mitotic stability of the transchromosomes in a heterologous genetic background. We have also produced chimeric mice carrying mardel(10) or NC-MiC1. Both transchromosomes were detected as intact episomal entities in a variety of adult chimeric mouse tissues including hemopoietic stem cells. Genes residing on these transchromosomes were expressed in the different tissues tested. Meiotic transmission of both transchromosomes in the chimeric mice was evident from the detection of DNA from these chromosomes in sperm samples. In particular, germ line transmission of NC-MiC1 was demonstrated in the F1 embryos of the chimeric mice. Variable (low in mardel(10)- or NC-MiC1-containing embryonic stem cells and chimeric mouse tissues and relatively high in NC-MiC1-containing F1 embryos) levels of missegregation of these transchromosomes were detected, suggesting that they are not optimally predisposed to full mitotic regulation in the mouse background, particularly during early embryogenesis. These results provide promising data in support of the potential use of neocentromere-based human marker chromosomes and minichromosomes as a tool for the study of centromere, neocentromere, and chromosome biology and for gene therapy studies in a mouse model system. They also highlight the need to further understand and overcome the factors that are responsible for the definable rates of instability of these transchromosomes in a mouse model. Centromeres are chromosomal loci that ensure the correct segregation of chromosomes and inheritance of genetic information. In humans, the centromeres consist of the 171-bp α-satellite DNA that is tandemly repeated up to several megabases in size. In contrast, human neocentromeres belong to a new class of centromeres that are devoid of α-satellite DNA formed epigenetically following cytogenetic rearrangements (reviewed in Ref. 1Amor D.J. Choo K.H. Am. J. Hum. Genet. 2002; 71: 695-714Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). These neocentromeres associate with all of the functionally essential centromere proteins and heterochromatin proteins to form active kinetochores conferring full mitotic stability (2Saffery R. Irvine D.V. Griffiths B. Kalitsis P. Wordeman L. Choo K.H. Hum. Mol. Genet. 2000; 9: 175-185Crossref PubMed Scopus (143) Google Scholar, 3Craig J.M. Wong L.H. Lo A.W. Earle E. Choo K.H. EMBO J. 2003; 22: 2495-2504Crossref PubMed Scopus (23) Google Scholar). The first described case of a neocentromere was found at the q25 region of a chromosome 10-derived marker chromosome mardel(10) (4Voullaire L.E. Slater H.R. Petrovic V. Choo K.H. Am. J. Hum. Genet. 1993; 52: 1153-1163PubMed Google Scholar, 5du Sart D. Cancilla M.R. Earle E. Mao J.I. Saffery R. Tainton K.M. Kalitsis P. Martyn J. Barry A.E. Choo K.H. Nat. Genet. 1997; 16: 144-153Crossref PubMed Scopus (276) Google Scholar). Human engineered chromosomes (HECs) 1The abbreviations used are: HEC, human engineered chromosome; CHO, Chinese hamster ovary; NC-MiC, neocentromere-based minichromosomes; ES, embryonic stem; FISH, fluorescence in situ hybridization; DAPI, 4′,6-diamidino-2-phenylindole; CENP, centomere protein; RT, reverse transcription; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; GFP, green fluorescent protein; MMCT, Microcell-mediated chromosome transfer; FACS, fluorescence-activated cell sorter; Mb, megabase. are autonomously replicating entities that can function and segregate as stable episomal entities. Such HECs serve as a useful model system for the study of centromere and chromosome properties and as gene expression vectors to complement genetic deficiencies in human cells. Early studies in the construction of HECs have involved either the transfection of α-satellite DNA into human cells (6Ebersole T.A. Ross A. Clark E. McGill N. Schindelhauer D. Cooke H. Grimes B. Hum. 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Biotechnol. 1998; 16: 431-439Crossref PubMed Scopus (359) Google Scholar) or the use of telomere-associated chromosomal truncation to remove the arms of endogenous chromosomes to produce minichromosomes (13Yang J.W. Pendon C. Yang J. Haywood N. Chand A. Brown W.R. Hum. Mol. Genet. 2000; 9: 1891-1902Crossref PubMed Scopus (70) Google Scholar, 14Shen M.H. Yang J. Loupart M.L. Smith A. Brown W. Hum. Mol. Genet. 1997; 6: 1375-1382Crossref PubMed Scopus (53) Google Scholar, 15Loupart M.L. Shen M.H. Smith A. Chromosoma. 1998; 107: 255-259Crossref PubMed Scopus (9) Google Scholar, 16Heller R. Brown K.E. Burgtorf C. Brown W.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7125-7130Crossref PubMed Scopus (133) Google Scholar, 17Farr C.J. Bayne R.A. Kipling D. Mills W. Critcher R. Cooke H.J. EMBO J. 1995; 14: 5444-5454Crossref PubMed Scopus (110) Google Scholar). Another strategy has involved the amplification of pericentric satellite DNA followed by the breakage of chromosomes to produce satellite DNA-based HECs (18Csonka E. Cserpan I. Fodor K. Hollo G. Katona R. Kereso J. Praznovszky T. Szakal B. Telenius A. deJong G. Udvardy A. Hadlaczky G. J. Cell Sci. 2000; 113: 3207-3216Crossref PubMed Google Scholar, 19Kereso J. Praznovszky T. Cserpan I. Fodor K. Katona R. Csonka E. Fatyol K. Hollo G. Szeles A. Ross A.R. Sumner A.T. Szalay A.A. Hadlaczky G. Chromosome Res. 1996; 4: 226-239Crossref PubMed Scopus (45) Google Scholar). The discovery of neocentromeres has provided an alternative approach to the construction of HECs. We have described previously the production of mitotically stable neocentromere-based minichromosomes (NC-MiCs) through the telomere-mediated truncation of the mardel(10) chromosome (20Saffery R. Wong L.H. Irvine D.V. Bateman M.A. Griffiths B. Cutts S.M. Cancilla M.R. Cendron A.C. Stafford A.J. Choo K.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5705-5710Crossref PubMed Scopus (62) Google Scholar). These NC-MiCs are amenable to full sequence characterization to provide a well defined tool. In addition, we have examined the expression status of coding genes within our 10q25 neocentromere on the mardel(10) chromosome and demonstrated transcriptional competency within the CENP-A and CENP-H domains and throughout the domain of enhanced scaffold/matrix attachment region, indicating that the process of neocentromere formation that results in the assembly of highly specialized centromeric chromatin has no measurable effect on gene expression (21Saffery R. Sumer H. Hassan S. Wong L.H. Craig J.M. Todokoro K. Anderson M. Stafford A. Choo K.H. Mol. Cell. 2003; 12: 509-516Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Mice containing HECs or human chromosomal fragments have been generated to provide animal models for HEC studies; however, little is known regarding the stability and expression status of these exogenous chromosomes in the different mouse tissues (22Co D.O. Borowski A.H. van der Leung J.D. Kaa J. Hengst S. Platenburg G.J. Pieper F.R. Perez C.F. Jirik F.R. Drayer J.I. Chromosome Res. 2000; 8: 183-191Crossref PubMed Scopus (71) Google Scholar, 23Hernandez D. Mee P.J. Martin J.E. Tybulewicz V.L. Fisher E.M. Hum. Mol. Genet. 1999; 8: 923-933Crossref PubMed Scopus (51) Google Scholar, 24Kuroiwa Y. Tomizuka K. Shinohara T. Kazuki Y. Yoshida H. Ohguma A. Yamamoto T. Tanaka S. Oshimura M. Ishida I. Nat. Biotechnol. 2000; 18: 1086-1090Crossref PubMed Scopus (122) Google Scholar, 25Shen M.H. Mee P.J. Nichols J. Yang J. Brook F. Gardner R.L. Smith A.G. Brown W.R. Curr. Biol. 2000; 10: 31-34Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 26Shinohara T. Tomizuka K. Takehara S. Yamauchi K. Katoh M. Ohguma A. Ishida I. Oshimura M. Chromosome Res. 2000; 8: 713-725Crossref PubMed Scopus (46) Google Scholar, 27Tomizuka K. Yoshida H. Uejima H. Kugoh H. Sato K. Ohguma A. Hayasaka M. Hanaoka K. Oshimura M. Ishida I. Nat. Genet. 1997; 16: 133-143Crossref PubMed Scopus (223) Google Scholar, 28Tomizuka K. Shinohara T. Yoshida H. Uejima H. Ohguma A. Tanaka S. Sato K. Oshimura M. Ishida I. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 722-727Crossref PubMed Scopus (176) Google Scholar, 29Voet T. Vermeesch J. Carens A. Durr J. Labaere C. Duhamel H. David G. Marynen P. Genome Res. 2001; 11: 124-136Crossref PubMed Scopus (50) Google Scholar). In this study, we have generated transgenic mice containing a neocentromere-based mardel(10) or NC-MiC and studied the in vivo stability and gene expression status of these chromosomes in a range of tissues and cell types. Cell Culture and Transfection—HT1080 cell line containing NC-MiC1 and CHOK1-derived ZB30 cells containing mardel(10) tagged with zeocin resistance gene were cultured in DMEM (Invitrogen) with 10% FCS or KAO-modified Hams' medium, respectively, as described previously (20Saffery R. Wong L.H. Irvine D.V. Bateman M.A. Griffiths B. Cutts S.M. Cancilla M.R. Cendron A.C. Stafford A.J. Choo K.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5705-5710Crossref PubMed Scopus (62) Google Scholar). Zeocin (Invitrogen) was added into both cultures at a final concentration of 100 μg/ml. Mouse embryonic stem cell line ES129.1 was cultured in DMEM with 15% heat-inactivated FCS and 103 units/ml leukemic inhibiting factor (Chemicon International) and 0.1 mm β-mercaptoethanol. The mouse ES129.1 cell line expressing green fluorescent protein (GFP) was generated by the transfection of a plasmid pEF-GCF-C1 containing the GFP and neomycin-resistant genes. 5 × 107 cells were electroporated (1.2 kV/25 microfarads, Bio-Rad Gene Pulser electroporator) with 15 μg of linearized construct. Cells were plated 24–48 h posttransfection, and 200 μg/ml neomycin G418 (Invitrogen) was added into the culture for selection of positive clones. Microcell-mediated Chromosome Transfer (MMCT)—Microcell fusion was carried out to transfer mardel(10) and NC-MiC1 tagged with zeocin resistance gene from the CHOK1 and HT1080 respective backgrounds into neomycin-resistant mouse ES129.1 cells expressing GFP (ES129.1GFP). Log-phase donor ZB30 cells were arrested in colcemid (Invitrogen) for 48 h and resuspended in Percoll/serum-free DMEM (1:1) supplemented with 20 μg/ml cytochalasin B (Sigma). The cell suspension was then subjected to centrifugation at 18,000 rpm for 90 min at 32 °C. Both bands of cell mixture were pelleted, washed with serum-free DMEM, and filtered through isopore membranes of 30, 8, and 5 μm (Millipore Corp.). Microcells were then fused with recipient neomycin-resistant ES-GFP cells by the addition of 50% polyethylene glycol (Roche Applied Science) for 2 min at room temperature. After incubation, cells were rinsed and cultured overnight in ES medium containing 20% FCS followed by the addition of selection (250 μg/ml neomycin and 100 μg/ml Zeocin) 24 h later. Fluorescence in Situ Hybridization (FISH)—Standard procedures were followed for FISH analysis. Microtubule-depolymerizing agent colcemid was added at 10 μm for 1 h before the harvesting of the cells. These cells were then subjected to hypotonic treatment in 0.075 m of KCl followed by fixation in 3:1 methanol/acetic acid prior to spreading onto slides. The slides were then dehydrated in an ethanol series, denatured in 70% formamide/2× SSC at 70 °C, and hybridized at 37 °C overnight with the relevant labeled probes. After three washes in 0.1× SSC at 60 °C, the probes were detected with fluorescence-conjugated reagents according to the manufacturer's instruction. Images were collected using a fluorescence microscope linked to a CCD camera system. For reverse painting of NC-MiC1, total genomic DNA from ESGFPNC-MiC1 was subjected to Alu-PCR amplification (30Liu P. Siciliano J. Seong D. Craig J. Zhao Y. de Jong P.J. Siciliano M.J. Cancer Genet. Cytogenet. 1993; 65: 93-99Abstract Full Text PDF PubMed Scopus (92) Google Scholar) followed by standard FISH analysis. Blastocyst Injection and Generation of Chimeric Mice—ES129.1GFP cell lines containing mardel(10) (ESGFPmar(10)-1) and NC-MiC1 (ES-GFPNC-MiC1-2) were injected into C57BL/6 blastocysts by standard procedures (31Bradley A. Ramirez-Solis R. Zheng H. Hasty P. Davis A. CIBA Found. Symp. 1992; 165: 256-276PubMed Google Scholar). The injected blastocysts were then transferred into recipient pseudopregnant mice. Chimeric mice were selected by coat color. Chimeric mice were also crossed with C57BL/6 mice to generate embryos and mice containing germ line transmitted mardel(10) or NC-MiC1. Tissue Collection and Genotyping of Chimeric Mice and Embryo— Tissues collected from chimeric mice were subjected to DNA isolation using QIAamp tissue purification kit (Qiagen) according to manufacturers' protocols. In addition, certain tissues including lung, kidney, skin, and tail were also cultured in DMEM containing 10% FCS for further analysis. The sperm samples were harvested from the uteri of the female C57BL/6 mice that had been mated with the chimeric mice generated as described above (32Mann G.B. Fowler K.J. Grail D. Dunn A.R. J. Reprod. Fertil. 1993; 99: 505-512Crossref PubMed Scopus (9) Google Scholar). To purify the hemopoietic stem cells, bone marrow was collected from mice (pretreated with 150 mg/kg body mass fluorouracil for 4 days) followed by washing with 1× phosphate-buffered saline and lysis on ice for 10 min (lysis solution 0.83% NH4Cl and 0.084% NaHCO3). The cells were then stained with R-phycoerythrin-conjugated rat anti-mouse Ly-6A/E (Sca-1) monoclonal antibody prior to sorting by FACS. The Ly-6A/E (Sca-1)-positive hemopoietic stem cells isolated by FACS were then subjected to another round of sorting to enrich the ES129.1GFP-derived cells. Cells collected were also cultured in the presence of 50 ng/ml stem cell factor, 10 ng/ml interleukin 3, 6 ng/ml interleukin 6, and 100 ng/ml FLT3 (Chemicon International). Individual F1 embryos were dissected from either the oviducts at 2.5 and 3.5 days or the implantation site at 9.5 days post coitum and washed in 1× phosphate-buffered saline. They were then subjected to either DNA purification for PCR analysis or incubation in DMEM medium containing 10 μm colcemid for 3 h prior to fixation onto slides for FISH analysis. PCR analysis was carried out on samples using the standard techniques. Primer sets neostart 1 (5′-ATGATTGAACAAGATGGATTGCAC-3′) and neocodR1 (5′-TGAGATGACAGGAGATCCTGC-3′) were used to detect the presence of ES129.1GFP-derived cells within tissues. For the identification of mardel(10) or NC-MiC1 in tissues or embryos, two primer sets were used (69k10F3, 5′-TTTGCTCACTAGCTGTCTCCTCAT-3′, with 69k10R6, 5′-GATCATTACCCAGACTCTGACCATT-3′, and 69k10F4, 5′-AGTTATGGAACTCACAAGACAGGAC-3′, with 69k10R7, 5′-ACATGATGTGGTAGTTGAGTTCACA-3′). RNA Purification and RT-PCR—Total RNA was isolated using TRIzol (Invitrogen) according to the manufacturer's instructions. RT-PCR was carried out using Titan one-step PCR kit (Roche Applied Science) according to the manufacturer's protocols or a two-step procedure using cDNA prepared using TAQman reverse transcription reagents (Applied Biosystems) followed by standard PCR. Sequence-tagged Site PCR Analysis—Initial characterization of NC-MiC1 was carried out using sequence-tagged site PCR analysis and standard PCR procedures using markers and primer pairs listed in Supplementary Table I. Characterization of NC-MiC1—We have previously produced a somatic hybrid cell line (designated ZB30) containing mardel(10) tagged with a zeocin resistance gene in a Chinese hamster ovary background (20Saffery R. Wong L.H. Irvine D.V. Bateman M.A. Griffiths B. Cutts S.M. Cancilla M.R. Cendron A.C. Stafford A.J. Choo K.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5705-5710Crossref PubMed Scopus (62) Google Scholar). The transfer of the mardel(10) chromosome via MMCT into human HT1080 cells followed by telomere-associated chromosome truncation resulted in a number of lines containing truncated minichromosome derivatives of mardel(10) (20Saffery R. Wong L.H. Irvine D.V. Bateman M.A. Griffiths B. Cutts S.M. Cancilla M.R. Cendron A.C. Stafford A.J. Choo K.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5705-5710Crossref PubMed Scopus (62) Google Scholar). A cell line, NC-MiC1 (zeoR), was identified by FISH as carrying a small minichromosome containing the 10q25-derived neocentromere region and the zeocin resistance gene from a separate region of the mardel(10) chromosome formed through unknown rearrangements during the MMCT procedure (Fig. 1a). Reverse painting demonstrated that this minichromosome was derived solely from DNA of the 10q25 and 10p15 regions of chromosome 10 (Fig. 1c). As we had previously characterized the 10q25 region contained in this minichromosome (20Saffery R. Wong L.H. Irvine D.V. Bateman M.A. Griffiths B. Cutts S.M. Cancilla M.R. Cendron A.C. Stafford A.J. Choo K.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5705-5710Crossref PubMed Scopus (62) Google Scholar), sequence-tagged site PCR analysis was performed to determine the content of the 10p15-derived DNA of this minichromosome. A total of 47 primer pairs spanning 15 Mb of the 10p region (Supplementary Table I) were used in amplification experiments on ESGFPNC-MiC1 genomic DNA that contained NC-MiC1 alone in a mouse ES background (see below). Of these primer sets, five produced positive signals in the PCR amplification. BAC clones in this region were then used in FISH experiments to directly visualize the 10p15 DNA content of NC-MiC1 (Fig. 1, b–c). From these combined analyses, the structure of NC-MiC1 was determined and the total size was calculated as ∼3.5 Mb. Mardel(10) and NC-MiC1 Remain Structurally Intact and Stable following Transfer into Mouse ES Cells—In this study, both ZB30 and HT1080-NC-MiC1 lines were used as donors for MMCT into mouse embryonic stem cell line ES129.1GFP expressing GFP (see “Experimental Procedures”). Thirteen positive fusion cell lines containing mardel(10) and seven containing NC-MiC1 were isolated. One cell line containing mardel(10) (ESGF-Pmar(10)-1) and another containing NC-MiC1 (ESGFPNC-MiC1-2) were analyzed further. Mitotic stability of ESGFPmar(10)-1 over 80 cell divisions was 90% with selection and 80% without selection, suggesting that mardel(10) was largely stable but carried a very low rate (Table I). All of the other cell lines containing mardel(10) showed similar mitotic stability (with average loss rates of 0.15 and 0.25%/division with and without selection). NC-MiC1 was found to be slightly less stable in mouse ES 129.1 cells with retention rates of 70 and 55% with and without selection after 80 cell divisions, respectively. Likewise, all of the other NC-MIC1 containing cell lines were relatively similar in terms of their mitotic stability with the exception of two clones that demonstrated some degree of DNA amplification (with retention rates ranging from 50 to 75% after 80 divisions). Immunofluorescence using CREST6 autoimmune anticentromere serum (5du Sart D. Cancilla M.R. Earle E. Mao J.I. Saffery R. Tainton K.M. Kalitsis P. Martyn J. Barry A.E. Choo K.H. Nat. Genet. 1997; 16: 144-153Crossref PubMed Scopus (276) Google Scholar) and specific antiserum to centromere proteins CENP-A and CENP-E was positive on both mardel(10) and NC-MIC1 (Figs. 2 and 3). Detailed FISH analysis using human COT1, total genomic mouse DNA, mouse centromeric major and minor satellite DNAs, and various BAC probes at and surrounding the 10q25 neocentromere demonstrated that both the mardel(10) and NC-MiC1 remained structurally intact following MMCT transfer and had not acquired any mouse genomic sequences including centromeric elements (Figs. 2 and 3). Thus, notwithstanding the low measurable loss rate, the results demonstrate the overall function of human neocentromeres to support mitotic stability of intact mardel(10) and NC-MiC1 minichromosome in the mouse ES cell background.Table IStability of ESGFPmar(10)-1 and ESGFPNC-MiC1-2 ESGFPmar(10)-1 and ESGFPNC-MiC1-2 were cultured for 80 divisions in the presence (100 μg/ml) or absence of zeocin. The cells were harvested at various intervals to determine the stability of mardel(10) or NC-MiC1. Retention rates of 80–90% and 55–70% were observed for the respective marker chromosomes after 80 cell divisions with and without selection, implying a small loss rate over time in the mouse ES cell background.Cell linePassage no.Division no.Drug selectionNo. of cells scoredPercentage of retentionLoss per division%ESGFPmar(10)-1520Zeocin2020/20 (100)01040Zeocin2019/20 (95)0.131560Zeocin2018/20 (90)0.172080Zeocin2018/20 (90)0.13ESGFPmar(10)-15202020/20 (100)010402018/20 (90)0.2515602017/20 (85)0.2520802016/20 (80)0.25ESGFPNC-MiC1-2520Zeocin2020/20 (100)01040Zeocin2017/20 (85)0.381560Zeocin2015/20 (75)0.422080Zeocin2014/20 (70)0.38ESGFPNC-MiC1-25202020/20 (100)010402015/20 (75)0.6315602013/20 (65)0.5820802011/20 (55)0.56 Open table in a new tab Fig. 3Microcell-mediated chromosome transfer of NC-MiC-1 into mouse ES129.1GFP cells. I, FISH analysis of ESGFPNC-MiC1-2 cell line using 10q25 neocentromere-specific and flanking BAC probes (20Saffery R. Wong L.H. Irvine D.V. Bateman M.A. Griffiths B. Cutts S.M. Cancilla M.R. Cendron A.C. Stafford A.J. Choo K.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5705-5710Crossref PubMed Scopus (62) Google Scholar). NC-MiC1 is indicated by an arrow. a, i–iv, combined and split images for green, red, and DAPI, respectively. FISH shows the presence of 10q25 neocentromere-specific BAC probe B153g5 (ii, green) and the absence of distant p′ arm BAC B10k1 (iii, red). b, i–iv, combined and split images for green, red, and DAPI, respectively. FISH using neocentromere-specific probe E8 (ii, green) and human cot1 DNA (iii, red) shows the presence of NC-MiC1 and the absence of other human chromosomes in ESGFPNC-MiC1-2. II, FISH analysis of ESGFPNC-MiC1-2 using mouse DNA probes. NC-MiC1 is indicated by an arrow. i–iii, combined image and split images for red and green, respectively. a, FISH using BAC probe B153g5 (ii, green) and mouse centromeric major satellite DNA (iii, red), showing the absence of mouse major satellite in NC-MiC1 in ESGFPNC-MiC1-2. b, FISH using B153g5 (ii, green) and mouse centromeric minor satellite DNA (iii, red), showing the absence of mouse minor satellite in NC-MiC1 in ESGFPNC-MiC1-2. c, FISH using B153g5 (ii, green) and mouse genomic DNA paint (iii, red), showing the absence of mouse genomic DNA in NC-MiC1 in ESGFPNC-MiC1-2. d–e, FISH and immunofluorescence analysis of NC-MiC1. Green and red denote NC-specific probe E8 and centromere-specific antibodies including CREST6 (diii) and CENP-A (eiii), respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Chimeric Mice Retained Mardel(10) and NC-MiC1 Episomally and Relatively Stably in Many Tissue Types—Microinjection of neomycin-resistant ESGFPmar(10)-1 or ESGFPNC-MIC1-2 into mouse blastocysts followed by reimplantation into foster female mice resulted in 19 and 65 high grade chimeric mice, respectively. An analysis of tissue samples from adult mice by PCR using human chromosome 10q25-specific primers demonstrated the presence of both mardel(10) and NC-MiC1 in a variety of tissues (examples from two mice for each of mardel(10) and NC-MiC1 are shown in Tables II and III). In total, 85.7 and 84.6% chimeric mice were found to be positive for mardel(10) and NC-MiC1, respectively. Of the positive chimeric mice, we asked the question of what percentage of ES129.1GFP-derived tissues had retained the transchromosomes. The presence of parental ES129.1GFP cells was identified by PCR using primers corresponding to a part of the neomycin resistance gene that was present in these cells. The PCR results (some examples shown in Tables II and III) indicated that 74.6 and 69.5% ES129.1GFP-containing tissues were positive for mardel(10) and NC-MiC1, respectively. Together, the above analyses indicated ∼15% loss rate for the transchromosomes during chimeric mouse production and 25–30% loss rate within the ES cell-positive tissues of the transchromosomal mice.Table IIScreening of various tissues for the presence of mardel(10) in chimeric mouse PL (A) and CH (B) by PCR using specific primers to mardel(10) ES129.1 denotes PCR detection of neomycin resistance gene that was present in ES129.1 GFP-derived tissues. +ve and -ve denote the presence and absence of mardel(10) or the neomycin resistance gene in a particular tissue, respectively.(A) Chimeric mouse PLTissuesMardel(10)ES129.1Left lung+ve+veRight lung-ve+veLeft kidney+ve+veRight kidney-ve-veThymus-ve+veCecum+ve+veSmall intestine-ve-veLarge intestine-ve+veHeart+ve+veBrain+ve+veStomach-ve-vePale back skin+ve+veSternum+ve+veAdrenal-ve-veUterus-ve-veOvary-ve-veTongue-ve-veEsophagus-ve-veSalivary gland-ve-veLiver+ve+veBone marrow+ve+veSpleen+ve+vePancreas-ve+veTail-ve-veLeft leg-ve-veRight leg-ve+ve(B) Chimeric mouse CHTissuesMardel(10)ES129.1Left lung+ve+veRight lung-ve+veLeft kidney+ve+veRight kidney+ve+veThymus-ve-veCecum+ve+veSmall intestine-ve+veLarge intestine-ve-veHeart+ve+veBrain-ve-veStomach-ve-vePale back skin+ve+veSternum-ve-veAdrenal+ve+veUterus+ve+veOvary-ve+veTongue-ve+veEsophagus-ve-veSalivary gland-ve+veLiver+ve+veBone marrow+ve+veSpleen+ve+vePancreas+ve+veTail+ve+veLeft leg-ve-veRight leg-ve-ve Open table in a new tab Table IIIScreening of various tissues for presence of NC-MiC1 in chimeric mouse THO and JN by PCR using specific primers to NC-MiC1 ES129.1 denotes PCR detection of neomycin resistance gene that was present in ES129.1GFP-derived tissues. +ve and -ve denote the presence and absence of mardel(10) or the neomycin resistance gene in a particular tissue, respectively.(A) Chimeric mouse THOTissuesNC-MiC1ES129.1Left lung+ve+veRight lung+ve+veLeft kidney-ve-veRight kidney+ve+veSmall intestine-ve-veLarge intestine-ve-veHeart+ve+veBrain+ve+veStomach+ve+veThymus+ve+veAdrenal-ve+veTissuesNC-MiC1ES129.1Testis+ve+veEsophagus-ve-veLiver+ve+veBone marrow+ve+veSpleen+ve+veTail+ve+veSkeletal muscle+ve+veSkin+ve+veLeft eye+ve+veRight eye-ve-ve(B) Chimeric mouse JNTissuesNC-MiC1ES129.1Left lung+ve+veRight lung+ve+veLeft kidney-ve+veRight kidney+ve+veSmall intestine-ve+veLarge intestine+ve+veHeart-ve-veBrain-ve+veStomach+ve+veThymus-ve+veAdrenal+ve+veUterus-ve-veOvary-ve+veEsophagus-ve+veLiver+ve+veBone marrow+ve+veSpleen-ve+vePancreas+ve+veTail-ve-veLeft eye+ve+veSkeletal muscle+ve+ve Open table in a new tab Tissues such as skin, kidney, lung, and tail (other tissues were not viable in cultures) were collected from the transchromosomal chimeras for FISH analysis to determine the structural status of the introduced chromosomes. A combination of human and mouse cot1 DNA, whole human chromosome 10 paint, and neocentromere-specific BACs were used as FISH probes. The presence" @default.
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• W2035554773 title "Analysis of Mitotic and Expression Properties of Human Neocentromere-based Transchromosomes in Mice" @default.
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