Abstract
Human neocentromeres are functional centromeres that are devoid of the typical human centromeric alpha-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.
Highlights
10-derived neocentric marker chromosome, mardel(10), the functionally essential centromere proteins and heterochroand 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
Human engineered chromosomes (HECs)1 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
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
Summary
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 (20). Cells were plated 24 – 48 h posttransfection, and 200 g/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). The cell suspension was 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 M (Millipore Corp.). 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
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