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Cloning and construction of luciferase reporter plasmids.

MB Mickaël Bouvet
SV Stefanie Voigt
TT Takanobu Tagawa
MA Manuel Albanese
YC Yen-Fu Adam Chen
YC Yan Chen
DF Devin N. Fachko
DP Dagmar Pich
CG Christine Göbel
RS Rebecca L. Skalsky
WH Wolfgang Hammerschmidt
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Human 3′UTR sequences were amplified from genomic DNA by PCR with primers harboring Gateway-compatible extensions or restriction enzyme sites. RIG-I (DDX58), Viperin (RSAD2), IKKb (IKBKB), IRAK2, FYN, CBP, IRF9, JAK1, OAS2, JAK2, and MAP3K2 were cloned downstream of the Renilla luciferase (Rluc) in the modified psiCHECK-2 (Promega) plasmid p5522 using Gateway cloning technology (Fisher Scientific) (Fig. 3A to ​toEE and ​and4B4B to ​toG).G). To generate other 3′UTR luciferase reporters (Fig. S3), sequences were PCR amplified from genomic DNA of EBV-infected B cells and cloned into the XhoI and NotI sites downstream of Renilla luciferase in the psiCheck2 dual-luciferase reporter vector containing an expanded multiple-cloning site. When achievable, the entire 3′UTR was cloned for a given target. For longer 3′UTRs, a minimum of 1 kb containing the region predicted to be targeted by each miRNA was cloned. EBV miRNA expression vectors in pLCE are previously described (41).

Copyright and License information:  ©2021 Bouvet et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

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