Microcell-mediated chromosome transfer protocol

Microcell-mediated chromosome transfer (MMCT) was performed as described previously.^{7 (link),10 (link)} The tet-O HAC containing the loxP cassette was transferred from DT40 into CHO cells by MMCT technology. Briefly, microcells were prepared by centrifugation of 1 × 10⁹ DT40 cells attached on flasks (Nalge Nunc, Rochester, NY, USA) coated with poly-l-lysine (Sigma) and were fused to 1 × 10⁶ CHO cells by 47% polyethylene glycol 1000 (WAKO, Osaka, Japan). CHO hybrids were selected in the presence of 4 µg/ml of BS and collected for expansion. After construction of the tet-O-EGFP HAC vector by Cre/loxP recombination in CHO cells, the HAC was transferred from CHO into HT1080 cells via MMCT. The HT1080 hybrids were selected in the presence of 4 µg/ml of BS.

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Iida Y., Kim J.H., Kazuki Y., Hoshiya H., Takiguchi M., Hayashi M., Erliandri I., Lee H.S., Samoshkin A., Masumoto H., Earnshaw W.C., Kouprina N., Larionov V, & Oshimura M. (2010). Human Artificial Chromosome with a Conditional Centromere for Gene Delivery and Gene Expression. DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes, 17(5), 293-301.

Publication 2010

Cells Centrifugation Cho cells Chromosome Hybrids Lysine Poly Polyethylene glycol 1000 Recombination Vector

Corresponding Organization :

Other organizations : Tottori University, National Cancer Institute, Kazusa DNA Research Institute, Wellcome Centre for Cell Biology, University of Edinburgh

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Protocol cited in 12 other protocols

Variable analysis

independent variables

The transfer of the tet-O HAC containing the loxP cassette from DT40 into CHO cells by MMCT technology
The transfer of the tet-O-EGFP HAC vector from CHO into HT1080 cells via MMCT

dependent variables

Selection of CHO hybrids in the presence of 4 µg/ml of BS
Selection of HT1080 hybrids in the presence of 4 µg/ml of BS

control variables

Centrifugation of 1 × 10^9 DT40 cells attached on flasks coated with poly-L-lysine
Fusion of microcells to 1 × 10^6 CHO cells by 47% polyethylene glycol 1000

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