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Supplementary Materials and methods
DNA transfection
The DT40 hybrid cells retaining a single copy of a human X chromosome fragment was transfected with
the targeting/loxP construct pDYS-H3 by the electroporation of 1 x 107 cells with 25 g of NotI-linearized
plasmid at 25 F and 550 V in a 4 mm cuvette using a Gene Pulser (Bio-Rad, Hercules, CA). The cells
were resuspended in basic growth medium and aliquoted into four 96-well flat-bottomed microtiter plates
(Becton–Dickinson, Franklin Lakes, NJ). After 2 days, the cells were resuspended in selective medium
with 15 g/ml BS. About fourteen days later, drug-resistant colonies were picked up and expanded for the
following analyses. The DT40 cells containing the loxP site-targeted human X chromosome were
transfected with the targeting/chromosome truncation vector pDYS-tel2 and two days after transfection, the
transfected cells were selected with 0.5 g/ml puromycin (Sigma, St.Louis, MO) similarly as described
above. The Cre-expression vector, pBS185 (Invitrogen, Carlsbad, CA) was transfected into the CHO
hybrids containing the HAC vector and the modified X fragment using Lipofectamine 2000 reagent
(Invitrogen) according to the manufacturer’s protocol. After 24 hours of culture in basic growth medium,
the cells were cultured in medium containing 1 x HAT (Sigma) for selection. Fourteen days later,
drug-resistant colonies were picked up and expanded for further analyses described below.
Fluorescence in situ hybridization (FISH)
FISH analyses were performed using either fixed metaphase or interphase spreads of each cell hybrid using
the digoxigenin-labeled (Roche, Basel, Switzerland) human COT-1 DNA (Invitrogen) , the biotin-labeled
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BAC DNA (RP11-954B16), the biotin-labeled PGK-Puro plasmid DNA or the biotin-labeled CMV-BS
plasmid DNA essentially as described previously [26]. Chromosomal DNA was counterstained with
DAPI (Sigma). The images were captured using a microscope (Nikon, Tokyo, Japan) equipped with a
photometric CCD camera, digitally processed and visualized with the Argus system (Hamamatsu Photonics,
Hamamatsu, Japan).
Generation of chimeric mice containing the DYS-HAC
Chimera production was carried out as described previously [26]. Briefly, ES cells were injected into
8-cell stage embryos derived from jcl: MCH (ICR) mice (Crea Japan, Tokyo, Japan) and then were
transferred into pseudopregnant jcl: MCH (ICR) females. All animal experiments were approved by the
Institutional Animal Care and Use Committee of Tottori University.
Immunofluorescence staining
Serial transverse sections (10 m) of the muscles on glass slides (MATSUNAMI, Osaka, Japan) were
generated using a cryostat (Fisher Scientific, Pittsburgh, PA). The sections were blocked with 1% BSA in
PBS for 1hour at room temperature, and incubated with the first antibody MANDYS106 (diluted 1:4; a gift
from Dr. G. Morris, Keele University, UK) for 1 hour at room temperature. After a rinse, the secondary
antibody Alexa Fluor 555 goat anti-mouse conjugate (diluted 1:1000; Molecular Probes, Eugene, OR) was
applied for 1 hour at room temperature. The images were taken with a microscope (Carl Zeiss, Heidenheim,
Germany).