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Treatment of CHO cells with Taxol and reversine improves micronucleation and microcell-mediated chromosome transfer efficiency.

ABSTRACT: Microcell-mediated chromosome transfer is an attractive technique for transferring chromosomes from donor cells to recipient cells and has enabled the generation of cell lines and humanized animal models that contain megabase-sized gene(s). However, improvements in chromosomal transfer efficiency are still needed to accelerate the production of these cells and animals. The chromosomal transfer protocol consists of micronucleation, microcell formation, and fusion of donor cells with recipient cells. We found that the combination of Taxol (paclitaxel) and reversine rather than the conventional reagent colcemid resulted in highly efficient micronucleation and substantially improved chromosomal transfer efficiency from Chinese hamster ovary donor cells to HT1080 and NIH3T3 recipient cells by up to 18.3- and 4.9-fold, respectively. Furthermore, chromosome transfer efficiency to human induced pluripotent stem cells, which rarely occurred with colcemid, was also clearly improved after Taxol and reversine treatment. These results might be related to Taxol increasing the number of spindle poles, leading to multinucleation and delaying mitosis, and reversine inducing mitotic slippage and decreasing the duration of mitosis. Here, we demonstrated that an alternative optimized protocol improved chromosome transfer efficiency into various cell lines. These data advance chromosomal engineering technology and the use of human artificial chromosomes in genetic and regenerative medical research.

SUBMITTER: Uno N

PROVIDER: S-EPMC10403731 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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Treatment of CHO cells with Taxol and reversine improves micronucleation and microcell-mediated chromosome transfer efficiency.

Uno Narumi N Satofuka Hiroyuki H Miyamoto Hitomaru H Honma Kazuhisa K Suzuki Teruhiko T Yamazaki Kyotaro K Ito Ryota R Moriwaki Takashi T Hamamichi Shusei S Tomizuka Kazuma K Oshimura Mitsuo M Kazuki Yasuhiro Y

Molecular therapy. Nucleic acids 20230715

Microcell-mediated chromosome transfer is an attractive technique for transferring chromosomes from donor cells to recipient cells and has enabled the generation of cell lines and humanized animal models that contain megabase-sized gene(s). However, improvements in chromosomal transfer efficiency are still needed to accelerate the production of these cells and animals. The chromosomal transfer protocol consists of micronucleation, microcell formation, and fusion of donor cells with recipient cel ...[more]

PMID: 37547291

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Project description:BackgroundMicrocell-mediated chromosome transfer (MMCT) is a technique by which a chromosome(s) is moved from donor to recipient cells by microcell fusion. Polyethylene glycol (PEG) has conventionally been used as a fusogen, and has been very successful in various genetic studies. However, PEG is not applicable for all types of recipient cells, because of its cell type-dependent toxicity. The cytotoxicity of PEG limits the yield of microcell hybrids to low level (10-6 to 10-5 per recipient cells). To harness the full potential of MMCT, a less toxic and more efficient fusion protocol that can be easily manipulated needs to be developed.ResultsMicrocell donor CHO cells carrying a human artificial chromosome (HAC) were transfected with genes encoding hemagglutinin (H) and fusion (F) proteins of an attenuated Measles Virus (MV) Edmonston strain. Mixed culture of the CHO transfectants and MV infection-competent human fibrosarcoma cells (HT1080) formed multinucleated syncytia, suggesting the functional expression of the MV-H/F in the CHO cells. Microcells were prepared and applied to HT1080 cells, human immortalized mesenchymal stem cells (hiMSC), and primary fibroblasts. Drug-resistant cells appeared after selection in culture with Blasticidin targeted against the tagged selection marker gene on the HAC. The fusion efficiency was determined by counting the total number of stable clones obtained in each experiment. Retention of the HAC in the microcell hybrids was confirmed by FISH analyses. The three recipient cell lines displayed distinct fusion efficiencies that depended on the cell-surface expression level of CD46, which acts as a receptor for MV. In HT1080 and hiMSC, the maximum efficiency observed was 50 and 100 times greater than that using conventional PEG fusion, respectively. However, the low efficiency of PEG-induced fusion with HFL1 was not improved by the MV fusogen.ConclusionsEctopic expression of MV envelope proteins provides an efficient recipient cell-oriented MMCT protocol, facilitating extensive applications for studies of gene function and genetic corrections.

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Treatment of CHO cells with Taxol and reversine improves micronucleation and microcell-mediated chromosome transfer efficiency.

Publications

Treatment of CHO cells with Taxol and reversine improves micronucleation and microcell-mediated chromosome transfer efficiency.

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