SCR726
TagRFP-Simplicon®-Plasmid (E3L)
Anmeldenzur Ansicht organisationsspezifischer und vertraglich vereinbarter Preise
Alle Fotos(1)
About This Item
UNSPSC-Code:
12352207
eCl@ss:
32161000
NACRES:
NA.51
Empfohlene Produkte
Methode(n)
nucleic acid detection: suitable (RNA)
Versandbedingung
ambient
Allgemeine Beschreibung
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 SimpliconTM 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 TagRFP Simplicon Plasmid (E3L) was developed for the synthesis of TagRFP Simplicon RNA (E3L). The Simplicon TagRFP RNA may be used to determine optimal transfection conditions to express the self-replicating RNA in hard-to transfect somatic or primary cells. Simplicon TagRFP expresses a monomeric red (orange) fluorescent protein generated from the RFP of sea anemone Entacmaea quadricolor. TagRFP exhibits fluorescence with excitation/emission maxima at 555/584 nm respectively, and brightness that is nearly three times higher than mCherry7,8.
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 SimpliconTM 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 TagRFP Simplicon Plasmid (E3L) was developed for the synthesis of TagRFP Simplicon RNA (E3L). The Simplicon TagRFP RNA may be used to determine optimal transfection conditions to express the self-replicating RNA in hard-to transfect somatic or primary cells. Simplicon TagRFP expresses a monomeric red (orange) fluorescent protein generated from the RFP of sea anemone Entacmaea quadricolor. TagRFP exhibits fluorescence with excitation/emission maxima at 555/584 nm respectively, and brightness that is nearly three times higher than mCherry7,8.
Anwendung
Das Simplicon-TagRFP-Plasmid (E3L) wurde entwickelt, um die Beurteilung des Simplicon-Expressionssystems in Ihren Zelllinien zu ermöglichen. Es kann beispielsweise zur Bestimmung optimaler Transfektionsbedingungen verwendet werden, um die relevante selbstreplizierende RNA durch die Verwendung des Simplicon-Klonierungsvektors (E3L) (Art.-Nr. SCR724) in schwer zu transfizierenden somatischen oder primären Zellen zu exprimieren.
Lagerung und Haltbarkeit
TagRFP-SimpliconTM-Plasmid (E3L) (Art.-Nr. SCR726). Ein (1) Fläschchen mit 10 µl Plasmid (1 µg/µl). Bei -20 °C aufbewahren.
Sonstige Hinweise
Konzentration: Die Konzentration entnehmen Sie bitte dem chargenspezifischen Datenblatt.
Rechtliche Hinweise
SIMPLICON is a registered trademark of Merck KGaA, Darmstadt, Germany
Haftungsausschluss
Sofern in unserem Katalog oder anderen Begleitdokumenten unserer Produkte nicht anders angegeben, sind unsere Produkte nur für Forschungszwecke vorgesehen und nicht für andere Zwecke zu verwenden, einschließlich, jedoch nicht beschränkt auf unautorisierte kommerzielle Verwendung, zur In-vitro-Diagnostik, für Ex-vivo- oder In-vivo-Therapiezwecke oder jegliche Art der Einnahme oder Anwendung bei Menschen oder Tieren.
Lagerklassenschlüssel
12 - Non Combustible Liquids
Analysenzertifikate (COA)
Suchen Sie nach Analysenzertifikate (COA), indem Sie die Lot-/Chargennummer des Produkts eingeben. Lot- und Chargennummern sind auf dem Produktetikett hinter den Wörtern ‘Lot’ oder ‘Batch’ (Lot oder Charge) zu finden.
Besitzen Sie dieses Produkt bereits?
In der Dokumentenbibliothek finden Sie die Dokumentation zu den Produkten, die Sie kürzlich erworben haben.
Unser Team von Wissenschaftlern verfügt über Erfahrung in allen Forschungsbereichen einschließlich Life Science, Materialwissenschaften, chemischer Synthese, Chromatographie, Analytik und vielen mehr..
Setzen Sie sich mit dem technischen Dienst in Verbindung.