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テクニック
nucleic acid detection: suitable (RNA)
輸送温度
ambient
詳細
Simplicon is a novel system to effect immediate high sustained protein expression of multiple genes into transfected cells without the risk of genome integration. The technology employs a single, synthetic, polycistronic, self-replicating RNA based on the Venezuelan equine encephalitis (VEE) genome1,2,3,4. The Simplicon RNA contains only genes encoding the VEE RNA replication machinery while the structural proteins that are required to make an infectious particle have been removed and replaced with the transgenes of interest. The Simplicon RNA is a synthetic RNA generated from the SimpliconTM Cloning Vector (E3L) plasmid.
Introduction and replication of the Simplicon RNA is expected to elicit a strong interferon response in transfected cells. To suppress the IFN responses, a Vaccinia virus protein5, B18R, is used for the original Simplicon technology. Recently, we found that another Vaccinia virus protein5, E3L, also suppresses the IFN responses in Simplicon RNA expression. B18R neutralizes type I interferons by direct binding, while E3L inhibits the cytoplasmic signaling pathways of IFN responses. Therefore, B18R and E3L are both employed in the Simplicon Expression System and work collaboratively to suppress IFN responses. As a result, there is increased cell viability during RNA transfection and increased expression of the transgenes. The Simplicon Expression System works in human cells and is not expected to work in mouse cells. This is because the B18R does not effectively neutralize mouse interferon (IFN)-β.
One day after transfection of the Simplicon RNA, a spike in the levels of transgenes can be observed. The expression levels are maintained by addition of B18R, E3L and the selective agent, puromycin throughout the duration of the experiment. Over time, expression levels are expected to diminish and stabilize to 1/5 – 1/10 the levels initially observed and may be close to physiological levels after one week. Expression levels and duration may change depending upon the cell types, transgenes and media conditions used. The Simplicon technology has been successfully utilized for efficient human iPSC generation through the sustained expression of critical reprogramming factors3,4 and in the creation of cell lines that express and retain the metabolic activities of five cytochrome P450 enzymes6.
In the Simplicon Expression System, B18R and E3L are provided as a B18R-E3L RNA (Cat. No. SCR722) for the suppression of IFN responses at RNA transfection. For sustained transgene expression, recombinant B18R protein (Cat. No. SCR156 and SCR197) or B18R conditioned medium (B18R-CM) can be used. E3L is continuously provided from the Simplicon RNA itself. B18R-CM can be produced from B18R-E3L RNA or B18R RNA synthesized using the B18R-E3L plasmid (Cat. No. SCR727) or B18R plasmid (Cat. No. SCR728), respectively.
The Simplicon Cloning Vector (E3L) (Cat. No. SCR724) is designed for expression of the transgenes of interest as a Simplicon RNA. The vector encodes four non-structural replication complex proteins (nsPs) as a single ORF at the 5’ half of the RNA, and multiple cloning site, E3L and puromycin resistance gene at the 3’ half of the RNA. Simplicon Cloning Vector (E3L) can accommodate transgene cassettes up to a total size of 8,300 bp. The presence of E3L in the Simplicon RNA works to suppress the IFN responses for sustained expression of the Simplicon RNA. For suppression of IFN responses at RNA transfection, we recommend B18R-E3L RNA (SCR722) prepared from the B18R-E3L plasmid (SCR727).
Introduction and replication of the Simplicon RNA is expected to elicit a strong interferon response in transfected cells. To suppress the IFN responses, a Vaccinia virus protein5, B18R, is used for the original Simplicon technology. Recently, we found that another Vaccinia virus protein5, E3L, also suppresses the IFN responses in Simplicon RNA expression. B18R neutralizes type I interferons by direct binding, while E3L inhibits the cytoplasmic signaling pathways of IFN responses. Therefore, B18R and E3L are both employed in the Simplicon Expression System and work collaboratively to suppress IFN responses. As a result, there is increased cell viability during RNA transfection and increased expression of the transgenes. The Simplicon Expression System works in human cells and is not expected to work in mouse cells. This is because the B18R does not effectively neutralize mouse interferon (IFN)-β.
One day after transfection of the Simplicon RNA, a spike in the levels of transgenes can be observed. The expression levels are maintained by addition of B18R, E3L and the selective agent, puromycin throughout the duration of the experiment. Over time, expression levels are expected to diminish and stabilize to 1/5 – 1/10 the levels initially observed and may be close to physiological levels after one week. Expression levels and duration may change depending upon the cell types, transgenes and media conditions used. The Simplicon technology has been successfully utilized for efficient human iPSC generation through the sustained expression of critical reprogramming factors3,4 and in the creation of cell lines that express and retain the metabolic activities of five cytochrome P450 enzymes6.
In the Simplicon Expression System, B18R and E3L are provided as a B18R-E3L RNA (Cat. No. SCR722) for the suppression of IFN responses at RNA transfection. For sustained transgene expression, recombinant B18R protein (Cat. No. SCR156 and SCR197) or B18R conditioned medium (B18R-CM) can be used. E3L is continuously provided from the Simplicon RNA itself. B18R-CM can be produced from B18R-E3L RNA or B18R RNA synthesized using the B18R-E3L plasmid (Cat. No. SCR727) or B18R plasmid (Cat. No. SCR728), respectively.
The Simplicon Cloning Vector (E3L) (Cat. No. SCR724) is designed for expression of the transgenes of interest as a Simplicon RNA. The vector encodes four non-structural replication complex proteins (nsPs) as a single ORF at the 5’ half of the RNA, and multiple cloning site, E3L and puromycin resistance gene at the 3’ half of the RNA. Simplicon Cloning Vector (E3L) can accommodate transgene cassettes up to a total size of 8,300 bp. The presence of E3L in the Simplicon RNA works to suppress the IFN responses for sustained expression of the Simplicon RNA. For suppression of IFN responses at RNA transfection, we recommend B18R-E3L RNA (SCR722) prepared from the B18R-E3L plasmid (SCR727).
保管および安定性
Simplicon クローニングベクター （カタログ番号 SCR724）。10 µLのプラスミド（1 µg/µL）入りバイアル 1 本。 -20°Cで保存してください。
その他情報
濃度：データシートをご参照ください。
免責事項
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保管分類コード
12 - Non Combustible Liquids
適用法令
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Jan Code
SCR724:
試験成績書（COA）
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