Construction of Mouse A9 Clones Containing a Single Human Chromosome Tagged with Neomycin‐resistance Gene via Microcell Fusion

Abstract

Normal human fibroblasts (MRC‐5 or NTI‐4) were transfected with pSV2‐neo plasmid DNA. Fifty G418‐resistant fibroblast clones were isolated and independently fused to mouse A9 cells. The cell hybrids were selected and isolated in the medium containing G418 plus ouabaln. Since micronuclei were more efficiently induced in these hybrids compared to parental human fibroblasts by colcemid treatment, the transfer of neo‐tagged human chromosomes in the hybrids to mouse A9 cells was performed via microcell fusion. Two hundred A9 microcell hybrids were isolated and karyotyped. Among them, thirteen microcell clones, each containing a single human chromosome 1, 2, 5, 6, 7, 8, 10,11,12,15,18,19 or 20 were established. Isozyme analyses confirmed the presence of each human chromosome in these A9 microcell clones. The results of Southern blot and chromosomal in situ hybridization analyses indicate that the human chromosomes in these clones were tagged with pSV2‐ neo plasmid DNA.

The transfer of human artificial chromosomes via cryopreserved microcells

“…The number of GFP-positive clone was counted for the calculation of transfer efficiency Cytotechnology (2013) 65:803-809 805 green fluorescent protein (EGFP) and the blasticidin (Bsd) genes. When the CHO 4H6.1M cells were treated with colcemid, micronuclei were formed in CHO cells (Koi et al 1989). The microcells were isolated from the treated CHO 4H6.1M cells by centrifugation.…”

Position of human chromosomes is conserved in mouse nuclei indicating a species-independent mechanism for maintaining genome organization

“…Various assays have previously demonstrated transcripts of human origin in mouse cells. For instance, expression of human specific isozymes was detected in A9 monochromosomal hybrid cells (Koi, et al 1989). Mouse-human hybrid cells in which the entire mouse chromosomes were retained but the human chromosomes were selectively lost expressed human antigens on the cell surface (Weiss, et al 1967).…”

Identification of a ≦600-kb region on human chromosome 1q42.3 inducing cellular senescence

“…Microcell-mediated chromosome transfer was performed as described previously (Koi et al, 1989). Briefly, A9 (neo#1) or A9(bsr#1) cells were incubated with 0.05 mg/ml of colcemid for 48 h in DMEM containing 20% CS for 48 h. Micronuclei induced in these cells were treated with 10 mg/ml of cytochalasin B, centrifuged, and sequentially filtered through 8, 5 and 3 mm polycarbonate filters (Nucleopore).…”

“…In total, 800 mg/ml of G418 (GIBCO) or 4 mg/ml of blasticidin S was added to isolate B16 cells containing the introduced chromosomes. The donor A9 (neo #1) and A9 (bsr#1) cells are derivatives of mouse L cells and carry a single copy of a normal human chromosome-1-tagged pSV2neo or pSV2bsr and were cultured in DMEM supplemented 10% CS and 800 mg/ml of G418 or 3 mg/ml of blasticidin S (Koi et al, 1989;Kugoh et al, 1999). Senescence locus on human chromosome 1 T Yawata et al…”

“…Mouse A9 hybrid (Koi et al, 1989) andB16-F10 cells (Fidler andNicolson, 1976) were cultured in DMEM (GIBCO) supplemented with 10% CS (Hyclone), 100 units/ ml penicillin G sodium, and 100 mg/ml of streptomycin sulfate (GIBCO) at 371C in humidified 5% CO 2 . B16-F10 cells were used as recipient cells for microcell fusion.…”