Exploitation of the interaction of measles virus fusogenic envelope proteins with the surface receptor CD46 on human cells for microcell-mediated chromosome transfer.
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.
Katoh
Motonobu
M
Chromosome Engineering Research Center, Tottori University, Yonago 683-8503, Japan.
Kazuki
Yasuhiro
Y
Kazuki
Kanako
K
Kajitani
Naoyo
N
Takiguchi
Masato
M
Nakayama
Yuji
Y
Nakamura
Takafumi
T
Oshimura
Mitsuo
M
D000818:Animals; D016466:CHO Cells; D002459:Cell Fusion / Q000379:methods*; D045744:Cell Line, Tumor; D022222:Chromosomes, Artificial, Human; D006224:Cricetinae; D003412:Cricetulus; D005822:Genetic Vectors; D006801:Humans; D008459:Measles virus; D051916:Membrane Cofactor Protein / Q000737:chemistry*; D014759:Viral Envelope Proteins / Q000737:chemistry*
D051916:Membrane Cofactor Protein; D014759:Viral Envelope Proteins
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