Transfection of Sendai virus F gene cDNA with mutations at its cleavage site and HN gene cDNA into COS cells induces cell fusion

Taira, Hideharu; Sato, Toshitsugu; Segawa, Hiroaki; Chiba, Masanori; Katsumata, Teizo; Iwasaki, Kentaro

doi:10.1007/bf01309734

Cited by 6 publications

(12 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The SeVdp vector was rescued by transient expression of the SeV Fmut, HN, and M (Cl.151) genes (driven by the SR␣ promoter derived from pcDL-SR␣) (18) in the packaging cells as described above and recovered into the culture supernatant after incubation at 32°C for 4 days. Fmut, a modified F gene for expressing the protease-susceptible SeV F protein, was generated as described (17). The supernatant was filtered through 0.45-m cellulose acetate filters and stored in small aliquots at Ϫ80°C.…”

Section: Methodsmentioning

confidence: 99%

Development of Defective and Persistent Sendai Virus Vector

Nishimura

Sano²,

Ohtaka

et al. 2011

Journal of Biological Chemistry

332

194

View full text Add to dashboard Cite

The ectopic expression of transcription factors can reprogram differentiated tissue cells into induced pluripotent stem cells. However, this is a slow and inefficient process, depending on the simultaneous delivery of multiple genes encoding essential reprogramming factors and on their sustained expression in target cells. Moreover, once cell reprogramming is accomplished, these exogenous reprogramming factors should be replaced with their endogenous counterparts for establishing autoregulated pluripotency. Complete and designed removal of the exogenous genes from the reprogrammed cells would be an ideal option for satisfying this latter requisite as well as for minimizing the risk of malignant cell transformation. However, no single gene delivery/expression system has ever been equipped with these contradictory characteristics. Here we report the development of a novel replication-defective and persistent Sendai virus (SeVdp) vector based on a noncytopathic variant virus, which fulfills all of these requirements for cell reprogramming. The SeVdp vector could accommodate up to four exogenous genes, deliver them efficiently into various mammalian cells (including primary tissue cells and human hematopoietic stem cells) and express them stably in the cytoplasm at a prefixed balance. Furthermore, interfering with viral transcription/replication using siRNA could erase the genomic RNA of SeVdp vector from the target cells quickly and thoroughly. A SeVdp vector installed with Oct4/Sox2/Klf4/c-Myc could reprogram mouse primary fibroblasts quite efficiently; ϳ1% of the cells were reprogrammed to Nanog-positive induced pluripotent stem cells without chromosomal gene integration. Thus, this SeVdp vector has potential as a tool for advanced cell reprogramming and for stem cell research. The generation of induced pluripotent stem (iPS)3 cells by reprogramming tissue cells with defined factors opened the door for realizing the medical application of patient-derived engineered stem cells (1). iPS cells were established originally by the ectopic expression of multiple transcription factors (e.g. Oct3/4, Sox2, Klf4, and c-Myc) using a retroviral vector (1). Since then, researchers have established iPS cells by several different approaches (and by their combination), including gene transfer, protein transduction, and treatment with chemical compounds (2). However, because of superior reproducibility and efficacy, ectopic expression of reprogramming factors by gene transfer is still the primary method of choice.Various lines of evidence indicate that efficient cell reprogramming requires the sustained and simultaneous expression of several (usually 4) exogenous factors for at least 10 -20 days (3). On the other hand, after reprogramming has been completed, these exogenous factors should be replaced promptly with their endogenous counterparts if the cells are to acquire autoregulated pluripotency (3). For this reason, retroviral and lentiviral vectors have been used preferentially; chromosomal insertion of the vector genome allow...

show abstract

Section: Methodsmentioning

confidence: 99%

Development of Defective and Persistent Sendai Virus Vector

Nishimura

Sano²,

Ohtaka

et al. 2011

Journal of Biological Chemistry

332

194

View full text Add to dashboard Cite

show abstract

“…Cell fusion induction by SeV glycoproteins requires the cleavage of inactive precursor F 0 to the active form complex, F 1 -F 2 , and co-expression of the HN protein on the surface of the same cells. 13) Here, COS-1 cells co-transfected with pSRD-F-EGFP and pSRD-HN were washed with PBS containing 0.1% trypsin 48 h after transfection and incubated for 5 h. Extensive cell fusion was observed in the cells expressing the F-EGFP and HN proteins, and was detected throughout the cytoplasm as diffuse green fluorescence (EGFP) and red fluorescence (HN) respectively (Supplemental Fig. 1B; see Biosci.…”

Section: Expression and Functional Analysis Of The F-egfp Fusion Proteinmentioning

confidence: 99%

Construction and Characterization of a Fluorescent Sendai Virus Carrying the Gene for Envelope Fusion Protein Fused with Enhanced Green Fluorescent Protein

Miyazaki

Segawa

Yamashita

et al. 2010

Bioscience, Biotechnology, and Biochemistry

View full text Add to dashboard Cite

“…For cell fusion to be caused by Sendai virus glycoproteins, cleavage of the inactive precursor F 0 to the active-form complex, F 1 -F 2 , is needed, as is expression of the HN protein on the surface of the same cells. 22) We therefore assessed the ability of mutant HN proteins to promote F proteinmediated cell fusion by a syncytium assay. We reported previously that cotransfection of mutant F, (Fcl), which has a cleavage site similar to that of virulent NDV F and HN cDNAs, causes cell fusion in COS-1 cells, and causes cell fusion in HeLa cells even more readily without trypsin treatment.…”

Section: Hng1mentioning

confidence: 99%

“…We reported previously that cotransfection of mutant F, (Fcl), which has a cleavage site similar to that of virulent NDV F and HN cDNAs, causes cell fusion in COS-1 cells, and causes cell fusion in HeLa cells even more readily without trypsin treatment. 22) Therefore, HeLa cells were cotransfected with wild-type and glycosylation-mutant HN cDNAs and Fcl cDNA. The expression plasmid pSRD-Fcl was constructed as described previously.…”

Section: Hng1mentioning

confidence: 99%

“…The expression plasmid pSRD-Fcl was constructed as described previously. 22) HeLa cells were washed with PBS 24 h after transfection andˆxed with methanol at room temperature for 10 min. They were then stained with Giemsa's staining solution (Nacalai Tesque, Kyoto) at room temperature for 10 min, washed three times with water, and air dried, and the nuclei were counted under a microscopy in Giemsastained cultures.…”

Section: Hng1mentioning

confidence: 99%

See 1 more Smart Citation