Author: Bederka, Lydia H.; Bonhomme, Cyrille J.; Ling, Emily L.; Buchmeier, Michael J.
Title: Arenavirus Stable Signal Peptide Is the Keystone Subunit for Glycoprotein Complex Organization Document date: 2014_10_28
ID: wbh06gzb_30
Snippet: Plasmids. Total RNA was isolated using Trizol from BHK-21 cells infected with LCMV Arm-4 at a multiplicity of infection (MOI) of 0.1 for 48 h at 37°C, 5% CO 2 . Viral glycoprotein cDNA was synthesized using a GP-specific primer (sequence provided upon request) flanked by XhoI recognition sequences using the GoScript reverse transcriptase as per the manufacturer's protocol and cloned into the pTargeT vector (Promega). Point mutations were introdu.....
Document: Plasmids. Total RNA was isolated using Trizol from BHK-21 cells infected with LCMV Arm-4 at a multiplicity of infection (MOI) of 0.1 for 48 h at 37°C, 5% CO 2 . Viral glycoprotein cDNA was synthesized using a GP-specific primer (sequence provided upon request) flanked by XhoI recognition sequences using the GoScript reverse transcriptase as per the manufacturer's protocol and cloned into the pTargeT vector (Promega). Point mutations were introduced using the site-directed QuikChange Lightning kit (Agilent Technologies), sequence verified using standard automated sequencing methods, and subcloned into the pCAGGS vector using the XhoI sites (53) . The HA epitope tag (YPYDVPDYA), as well as the replacement of the native stable signal peptide with the signal sequence from the influenza A hemagglutinin protein (MEKIVLLFAIVS-LVKS), was inserted into the GPC open reading frame using the overlap PCR technique (54) .
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