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Exploitation of the interaction of measles virus fusogenic envelope proteins with the surface receptor CD46 on human cells for microcell-mediated chromosome transfer.

Katoh M, Kazuki Y, Kazuki K, Kajitani N, Takiguchi M, Nakayama Y, Nakamura T, Oshimura M - BMC Biotechnol. (2010)

Bottom Line: Polyethylene glycol (PEG) has conventionally been used as a fusogen, and has been very successful in various genetic studies.Retention of the HAC in the microcell hybrids was confirmed by FISH analyses.Ectopic expression of MV envelope proteins provides an efficient recipient cell-oriented MMCT protocol, facilitating extensive applications for studies of gene function and genetic corrections.

View Article: PubMed Central - HTML - PubMed

Affiliation: Chromosome Engineering Research Center, Tottori University, Yonago 683-8503, Japan.

ABSTRACT

Background: Microcell-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.

Results: Microcell 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.

Conclusions: Ectopic 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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The MV fusogen is more efficient than PEG for microcell hybrid production. Microcells collected from 24 flasks (~5 × 106) were fractionated into 5 dosing amounts (2 × 105, 4 × 105, 8 × 105, 1 × 106, 2 × 106) and added to (MV), or fused with (PEG), 2 × 106 of HT1080 cells in a 60-mm dish. On the following day, fused cells were plated onto two 100-mm dishes. Following selection culture for 14 days, drug-resistant colonies were stained with Giemsa and photographed.
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Figure 5: The MV fusogen is more efficient than PEG for microcell hybrid production. Microcells collected from 24 flasks (~5 × 106) were fractionated into 5 dosing amounts (2 × 105, 4 × 105, 8 × 105, 1 × 106, 2 × 106) and added to (MV), or fused with (PEG), 2 × 106 of HT1080 cells in a 60-mm dish. On the following day, fused cells were plated onto two 100-mm dishes. Following selection culture for 14 days, drug-resistant colonies were stained with Giemsa and photographed.

Mentions: We next compared the efficiency of microcell fusion by MV fusogen with that by PEG. For this purpose, different amount of microcells were applied to the fixed number of recipient HT1080 cells. From 5 × 107 of CHO4H6.1M cells, ~5 × 106 of microcells were obtained. These microcells were aliquoted in five fractions with the ratio of 1,2,4,6,11, and applied to a fixed number (2 × 106) of recipient cells. As a control, the conventional fusion experiment using PEG was performed with the parental HAC donor cell line CHOkkpqG4. Microcell fusion was determined as the number of drug-resistant colonies following selection culture with Blasticidin (Figure 5). The fusion efficiency by MV fusogen was consistently, and at most 10 times, higher than that by PEG (Table 1). Whereas fusion induced by PEG was gradually increased in a microcell dose-dependent manner, fusion induced by the MV fusogen did not show dose dependency. Input over threshold amount of microcells with MV fusogen seemed to reduce the appearance of microcell hybrids.


Exploitation of the interaction of measles virus fusogenic envelope proteins with the surface receptor CD46 on human cells for microcell-mediated chromosome transfer.

Katoh M, Kazuki Y, Kazuki K, Kajitani N, Takiguchi M, Nakayama Y, Nakamura T, Oshimura M - BMC Biotechnol. (2010)

The MV fusogen is more efficient than PEG for microcell hybrid production. Microcells collected from 24 flasks (~5 × 106) were fractionated into 5 dosing amounts (2 × 105, 4 × 105, 8 × 105, 1 × 106, 2 × 106) and added to (MV), or fused with (PEG), 2 × 106 of HT1080 cells in a 60-mm dish. On the following day, fused cells were plated onto two 100-mm dishes. Following selection culture for 14 days, drug-resistant colonies were stained with Giemsa and photographed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2874513&req=5

Figure 5: The MV fusogen is more efficient than PEG for microcell hybrid production. Microcells collected from 24 flasks (~5 × 106) were fractionated into 5 dosing amounts (2 × 105, 4 × 105, 8 × 105, 1 × 106, 2 × 106) and added to (MV), or fused with (PEG), 2 × 106 of HT1080 cells in a 60-mm dish. On the following day, fused cells were plated onto two 100-mm dishes. Following selection culture for 14 days, drug-resistant colonies were stained with Giemsa and photographed.
Mentions: We next compared the efficiency of microcell fusion by MV fusogen with that by PEG. For this purpose, different amount of microcells were applied to the fixed number of recipient HT1080 cells. From 5 × 107 of CHO4H6.1M cells, ~5 × 106 of microcells were obtained. These microcells were aliquoted in five fractions with the ratio of 1,2,4,6,11, and applied to a fixed number (2 × 106) of recipient cells. As a control, the conventional fusion experiment using PEG was performed with the parental HAC donor cell line CHOkkpqG4. Microcell fusion was determined as the number of drug-resistant colonies following selection culture with Blasticidin (Figure 5). The fusion efficiency by MV fusogen was consistently, and at most 10 times, higher than that by PEG (Table 1). Whereas fusion induced by PEG was gradually increased in a microcell dose-dependent manner, fusion induced by the MV fusogen did not show dose dependency. Input over threshold amount of microcells with MV fusogen seemed to reduce the appearance of microcell hybrids.

Bottom Line: Polyethylene glycol (PEG) has conventionally been used as a fusogen, and has been very successful in various genetic studies.Retention of the HAC in the microcell hybrids was confirmed by FISH analyses.Ectopic expression of MV envelope proteins provides an efficient recipient cell-oriented MMCT protocol, facilitating extensive applications for studies of gene function and genetic corrections.

View Article: PubMed Central - HTML - PubMed

Affiliation: Chromosome Engineering Research Center, Tottori University, Yonago 683-8503, Japan.

ABSTRACT

Background: Microcell-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.

Results: Microcell 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.

Conclusions: Ectopic expression of MV envelope proteins provides an efficient recipient cell-oriented MMCT protocol, facilitating extensive applications for studies of gene function and genetic corrections.

Show MeSH
Related in: MedlinePlus