Prevention of PERV Infections in Pig to Human Xenotransplantation by the RNA Interference Silences Gene

The possibility of preventing the transmission of porcine endogenous retrovirus (PERV) to human cells using short interfering RNAs (siRNA) was investigated. The siRNA for the p30 of PERV gag region was cloned into pSUPER, the polymerase-III H1-RNA gene promoter. A green fluorescence protein (GFP) wa...

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Bibliographic Details
Published in:Journal of biochemistry (Tokyo) 2005-04, Vol.137 (4), p.503-508
Main Authors: Miyagawa, Shuji, Nakatsu, Shino, Nakagawa, Takatoshi, Kondo, Akihiro, Matsunami, Katsuyoshi, Hazama, Kenji, Yamada, Junko, Tomonaga, Keizo, Miyazawa, Takayuki, Shirakura, Ryota
Format: Article
Language:English
Subjects:
Animals
Animals, Genetically Modified
BFU
blue focus forming unit
Cells, Cultured
Endogenous Retroviruses - pathogenicity
GFP
green fluorescence protein
Humans
PEC
PERV
PERV-B
pig endothelial cell
pig endothelial cells
Porcine endogenous retrovirus
porcine endogenous retroviruses
pseudotype infection
Retroviridae Infections - prevention & control
Retroviridae Infections - transmission
RNA Interference
RNA, Small Interfering - therapeutic use
short interference RNA
siRNA
Swine
Transplantation, Heterologous - adverse effects
xenotransplantation
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Summary:The possibility of preventing the transmission of porcine endogenous retrovirus (PERV) to human cells using short interfering RNAs (siRNA) was investigated. The siRNA for the p30 of PERV gag region was cloned into pSUPER, the polymerase-III H1-RNA gene promoter. A green fluorescence protein (GFP) was also cloned into pSUPER to establish pSXGH. Pig endothelial cells (PEC) were transduced with the LacZ gene by pseudotype infection, and infected with PERV subtype B, resulting in the formation of PEC(LacZ)/PB. The PEC(LacZ)/PB was next transfected with pSXGH-siRNA. The expression of siRNA was provisionally checked by determining the level of expression of GFP. Culture supernatants of infected cells were then inoculated into HEK293 cells. The siRNA clearly destroyed the PERV infectivity of PEC(LacZ)/PB in both transient cell lines and stable clones. Moreover, the decreased levels of mRNA and gag protein were evidenced in the stable clones by real-time PCR and Western blotting, respectively. The final goal of our study was to establish a transgenic pig expressing the siRNA for PERV. The results suggest that siRNA represents a novel approach for controlling PERV infections in clinical xenotransplantation.
ISSN:0021-924X
1756-2651
DOI:10.1093/jb/mvi059