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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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Related in: MedlinePlus

Detection of chromosome transfer from the microcell to the donor cell. Recipient cells exposed to the microcells, and selected by Blasticidin treatment, were analyzed by fluorescence in situ hybridization to detect cells containing the transferred HAC. The HAC is delineated using an alphoid satellite probe derived from human chromosome 21, and is shown in red (arrow). The alphoid satellite probe detects endogenous chromosomes 13 and 21 (arrowhead), in addition to the HAC, due to their sequence similarity.
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Figure 4: Detection of chromosome transfer from the microcell to the donor cell. Recipient cells exposed to the microcells, and selected by Blasticidin treatment, were analyzed by fluorescence in situ hybridization to detect cells containing the transferred HAC. The HAC is delineated using an alphoid satellite probe derived from human chromosome 21, and is shown in red (arrow). The alphoid satellite probe detects endogenous chromosomes 13 and 21 (arrowhead), in addition to the HAC, due to their sequence similarity.

Mentions: FISH analysis was performed with the GFP expressing cells for the detection of the HAC transfer (Figure 4). Alphoid satellite probe derived from human chromosome 21 hybridized with endogenous chromsomes 21 and 13, due to their sequence similarity [14]. In addition to these endogenous chromosomes, the probe detected a minichromosome, which is not present in parental HT1080 cells (data not shown). Since the HAC was constructed by deleting almost all sequences from long and short arm of the chromosome 21, it retains alphoid satellite as substantial contents [14]. The estimated size of the HAC is less than 10 Mb, which corresponds to 1/5 of the original human chromosome 21. In comparison to the endogenous chromosome 21, the size of the minichromosome matched to the prediction. Obvious change in the karyotype of the host cells was not detected. These results demonstrated the introduction of the HAC by microcell fusion without disturbance of the host chromosomes.


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)

Detection of chromosome transfer from the microcell to the donor cell. Recipient cells exposed to the microcells, and selected by Blasticidin treatment, were analyzed by fluorescence in situ hybridization to detect cells containing the transferred HAC. The HAC is delineated using an alphoid satellite probe derived from human chromosome 21, and is shown in red (arrow). The alphoid satellite probe detects endogenous chromosomes 13 and 21 (arrowhead), in addition to the HAC, due to their sequence similarity.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Detection of chromosome transfer from the microcell to the donor cell. Recipient cells exposed to the microcells, and selected by Blasticidin treatment, were analyzed by fluorescence in situ hybridization to detect cells containing the transferred HAC. The HAC is delineated using an alphoid satellite probe derived from human chromosome 21, and is shown in red (arrow). The alphoid satellite probe detects endogenous chromosomes 13 and 21 (arrowhead), in addition to the HAC, due to their sequence similarity.
Mentions: FISH analysis was performed with the GFP expressing cells for the detection of the HAC transfer (Figure 4). Alphoid satellite probe derived from human chromosome 21 hybridized with endogenous chromsomes 21 and 13, due to their sequence similarity [14]. In addition to these endogenous chromosomes, the probe detected a minichromosome, which is not present in parental HT1080 cells (data not shown). Since the HAC was constructed by deleting almost all sequences from long and short arm of the chromosome 21, it retains alphoid satellite as substantial contents [14]. The estimated size of the HAC is less than 10 Mb, which corresponds to 1/5 of the original human chromosome 21. In comparison to the endogenous chromosome 21, the size of the minichromosome matched to the prediction. Obvious change in the karyotype of the host cells was not detected. These results demonstrated the introduction of the HAC by microcell fusion without disturbance of the host chromosomes.

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