Ethics statement
This study was approved by the Institutional Animal Care and Use Committee of Tottori University and the Recombinant DNA Experiment safety Committee of Tottori University (for performing recombinant DNA experiments). All experiments were carried out in compliance with the ARRIVE guidelines. All methods were carried out in accordance with relevant guidelines and regulations.
Cell culture
The mouse embryonic fibroblasts, mouse A9 cells, and whole cell hybrids were grown in Dulbecco’s modified Eagle’s medium (DMEM; Wako, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA). The DT40 hybrid cells were maintained in Roswell Park Memorial Institute (RPMI) medium 1640 (Wako, Tokyo, Japan) containing 10% FBS, 1% chicken serum (Gibco, ThermoFischer, Waltham, MA, USA), 50 μmol/L 2-mercaptoethanol, and the appropriate antibiotics. The DT40 hybrids containing a single copy of mouse chromosome 10 were produced by MMCT from mouse A9 hybrid cells containing NeoR-tagged mouse chromosome 10 and maintained with 1500 μg/mL G418 (Promega, Madison, WI, USA). The hypoxanthine phosphoribosyltransferase (Hprt)-deficient CHO (JCRB0218) hybrids containing a 10MAC1, 10MAC2 or 10MAC3 were maintained in Ham’s F-12 nutrient mixture (Wako, Tokyo, Japan) containing 10% FBS and 800 μg/mL G418. The parental mouse ES cell line, TT2F, and the microcell hybrid clone, TT2F (10MAC1), were maintained on mitomycin C (Sigma-Aldrich, St. Louis, MO, USA)-treated neomycin-resistant MEFs (Oriental Yeast, Tokyo, Japan) as feeder layers in DMEM with 18% FBS, 1 mM sodium pyruvate (Invitrogen, Carlsbad, CA, USA), 0.1 mM nonessential amino acids (Invitrogen, Carlsbad, CA, USA), 0.1 mM 2-mercaptoehtanol (Sigma-Aldrich, St. Louis, MO, USA), 2 mM l-glutamine (Invitrogen, Carlsbad, CA, USA), and 1,000 units/mL leukemia inhibitory factor (Funakoshi, Tokyo, Japan).
Construction of targeting vectors
The homologous regions on mouse chromosome 10 are described in Fig. 1. For constructing a telomere truncation vector, pBS-TEL/puro_10MAC, annealed sense and antisense EcoRI/AscI/EcoRI oligos were inserted into EcoRI site of pBS-TEL/Puro vector32. Then, a fragment (5.6 kb) of the mouse chromosome 10 region was amplified by PCR with AscI_m10T F2/BamHI_m10T R3 primers, digested with AscI/BamHI and sub-cloned into the AscI/BamHI sites of the pBS-TEL/Puro vector (pBS-TEL/puro_10MAC). The targeting vector, p10MAC1, for introducing EGFP/neo/loxP-3’HPRT was constructed as follows: Two 1.4 kb and 4.4 kb fragments for homologous arms corresponding to the mouse chromosome 10 pericentromeric region were amplified by PCR using the KpnI_m10 LA F/XhoI_m10 LA R primers (2.0 kb) and SalI_m10 RA F/ NotI_m10 RA R primers (4.4 kb), digested with KpnI/XhoI and SalI/NotI, and subcloned into pKO Scrambler V913 backbone vector (Lexicon Genetics, Woodlands, TX, USA). Then, the EGFP/neo/loxP-3’HPRT cassette from pVGNLH14, digested with SalI/AscI, was introduced into XhoI/AscI sites of the vector (p10MAC1). The targeting vector, p10MAC2, for introducing neo/5’HPRT-loxP was constructed as follows: PGKneo and 5’HPRT-loxP fragments were introduced into EcoRI site and AscI/ClaI sites of pKO Scrambler V907 backbone vector (Lexicon Genetics, Woodlands, TX, USA). Then, a 4.4 kb right arm was amplified by PCR using ClaI_m10 RA F/R primers and inserted into ClaI site of the vector (pN5’HLR). A 2.0 kb left arm was amplified by NotI_m10 LA F/SalI_m10 LA R primers and inserted into NotI/SalI sites of pN5’HLR (p10MAC2). The targeting vector, p10MAC3, for introducing EGFP/neo/5’HPRT-loxP was constructed as follows: CAG-EGFP flanked by insulator HS4 was inserted to NotI/SalI sites of pN5’HLR (pN5’HLER). Then, left arm was amplified by NotI_m10 LA F/SalI_m10 LA R primers and a 1.9 kb NotI/PspOMI-digested fragment was introduced to NotI site of the pN5’HLER (p10MAC3). Primer information is available in Supplementary Table S1.
Transfection of DT40 and CHO cells
The DT40 (mChr.10-neo) hybrid cells were transfected by electroporation of 1 × 107 cells with each NotI-linearized plasmid at 25 μF 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 96-well flat-bottomed microtiter plates with serial dilution (Becton–Dickinson, Franklin Lakes, NJ). Next day, the cells were resuspended in selective medium with 0.5 μg/ml puromycin or 1.5 mg/ml G418. Approximately fourteen days later, drug-resistant colonies were picked up and expanded for the following analyses. The CHO cells (2 × 106 cells) containing 10MAC1, 10MAC2 or 10MAC3 were transfected with pBS185 (CMV-Cre) and plasmids containing 5’HPRT-loxP (X6.1), loxP-3’HPRT-I-CAG-EGFP-I (X3.1-I-EGFP-I) or loxP-3’HPRT-I-EF1a-tdTomato-I (X3.1-I-tdTomato-I) by using 20 μL of Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacture’s instruction. After 24 h, cells were scaled up, and after 48 h, cultured in the medium containing 1 × HAT (Sigma-Aldrich, St. Louis, MO, USA) for selection. Fourteen days later, drug-resistant colonies were picked up and expanded for further analyses as described below.
MMCT
The 10MAC1, 10MAC2 and 10MAC3 vectors were transferred from the DT40 cells into the CHO hprt−/− cells using MMCT technology2. Briefly, microcells were prepared by centrifugation of DT40 cells attached to flasks (Nalge Nunc, Rochester, NY, USA) coated with poly-l-lysine followed by fusion with 1 × 106 CHO cells using 42% polyethylene glycol 1000 (Wako, Tokyo, Japan). CHO hybrids containing each 10MAC vector were selected with the media containing 800 μg/mL G418 and selected for expansion. CHO cells containing 10MAC1 without and with ecotropic EnvΔR expression were used as donor microcell hybrids in the PEG-MMCT and retro-MMCT as described previously18,33. mES XO ES9 hybrids containing 10MAC1 were selected with the media containing 150 μg/mL G418 and picked for expansion.
Genomic PCR analysis
Genomic DNA was extracted from cell lines using a genomic extraction kit (Gentra Systems, Minneapolis, MN, USA), and PCR was performed as follows. The screening primers for telomere truncation were m10 F6/PuroI, Gm8155 F/R, Iyd F/R and Plekhg1 F/R. The targeting with p10MAC1 was confirmed by using KpnI_m10 LA F/XhoI m10 LA R, m10 F1/EGFP-F and kj neo/m10 R2. The primers for detection of exact targeting with p10MAC2 were m10 F1/10MAC R1 and 10MAC F1/m10 R2. The targeted allele by p10MAC3 was analyzed by using m10 F1/EGFP-R and 10MAC F1/m10 R2. Contamination of DT40 cells after MMCT from DT40 cells to CHO cells were checked by using CENPH Fw/Rv. Contamination of CHO cells after MMCT from CHO cells to mES cells were checked by using Furin F/R. The recombination with Cre/loxP system on each 10MAC vector was detected by TRNS L1/R1. Primer sequences are described in Supplementary Table S1.
Fluorescence in situ hybridization (FISH) analysis
Preparation of metaphase chromosomes from exponentially growing cell culture was performed according to standard methods. FISH analyses were performed using the digoxigenin-labeled (Roche, Basel, Switzerland) mouse COT-1 DNA (Invitrogen, Carlsbad, CA, USA) or mouse minor satellite DNA, the biotin-labeled PGKpuro, pVGNLH1, X6.1, X3.1-I-EGFP-I, or X3.1-I-tdTomato-I plasmid DNA essentially as described previously33. Chromosomal DNA was counterstained with DAPI (Sigma-Aldrich, St. Louis, MO, US). Images were captured using an AxioImagerZ2 fluorescence microscope (Carl Zeiss, Oberkochen, Germany).
Generation of chimeric mice
Chimeric mice were produced from TT2F hybrids containing 10MAC1. Chimera production was performed as described previously33. mES cells were injected into 8-cell stage embryos derived from ICR mice (CLEA, Tokyo, Japan) and then transferred into pseudopregnant ICR females. Almost 100% coat-color chimeric mice were mated with ICR males to obtain Tc mice.
GFP image capture and FCM analysis of tissues and blood cells
Samples were collected from euthanized Tc mice containing 10MAC1 and their 10MAC1-negative counterpart with isoflurane after perfusion. Bright and GFP images of each tissue were obtained by M205 FA fluorescence stereo microscope (Leica Microsystems, Wetzlar, Germany) with NIS-Elements software (Nikon, Tokyo, Japan). The dissociated cells were treated with ammonium chloride solution for hemolysis and stained with anti-mouse B220/CD45R (Biolegend, San Diego, CA, USA), CD3 (Biolegend, San Diego, CA, USA), CD4 (Biolegend, San Diego, CA, USA) and CD8a (BD Biosciences, San Jose, CA, USA) antibodies conjugated with BV650, BV650, PE and PE-CF594, respectively. All staining samples (1 × 106 cells) were incubated at 4 °C for 30 min in 100 μl FCM buffer (HBSS with 5% FBS and 1 mM EDTA) (Gibco, ThermoFischer, Waltham, MA, USA). DAPI was added to the final suspension to exclude dead cells. Analyses were conducted using a CytoFLEX S (Beckman Coulter, Brea, CA, USA). Percentage of GFP-expressing cells in lymphocytes through generations was analyzed by Gallios (Beckman Coulter, Brea, CA, USA).
