Cloning and Mutagenesis of DHX36 Constructs

Full-length complementary DNA of wild-type HEK293 T-Rex Flp-In cells was used as template for cloning DHX36 into the pFRT/TO/FLAG/HA-Dest plasmid^{62 (link)} using the restriction enzymes BamHI (Thermo Fisher Scientific) and XhoI (Thermo Fisher Scientific). The so-generated pFRT-FlagHA-DHX36-iso1 was used to create the plasmids pFRT-FlagHA-DHX36-iso2, pFRT-FlagHA-DHX36-iso1-E335A, and pFRT-FlagHA-DHX36-iso2-E335A by site-directed mutagenesis. For this, primer with the desired mutation were designed and set in a PCR with the paternal plasmid and 2× Phusion PCR Master Mix (Thermo Fisher Scientific).
Reporter gene plasmids were generated by cloning DHX36 binding sites of the WAC and PURB mRNA 3′ UTRs as well as two non-targeted regions of the DDX5 mRNA 3′ UTR into the pcDNA5-FRT-GFP-mCherry-3pGW backbone^{63 (link)} (Addgene) using the commercial BP and LR clonase systems according to the manufacturer’s instructions (Thermo Fisher Scientific). Mutated version were generated by site-directed mutagenesis.

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Sauer M., Juranek S.A., Marks J., De Magis A., Kazemier H.G., Hilbig D., Benhalevy D., Wang X., Hafner M, & Paeschke K. (2019). DHX36 prevents the accumulation of translationally inactive mRNAs with G4-structures in untranslated regions. Nature Communications, 10, 2421.

Publication 2019

BamHI XhoI 2× Phusion PCR Master Mix

Binding sites Cells Complementary dna Ddx5 Mrna Mutation Plasmids Primer Reporter gene Restriction enzymes bamhi Site directed mutagenesis

Corresponding Organization :

Other organizations : University of Würzburg, University Hospital Bonn, National Institute of Arthritis and Musculoskeletal and Skin Diseases, University of Groningen, University Medical Center Groningen

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Variable analysis

independent variables

Restriction enzymes BamHI and XhoI used for cloning DHX36 into the pFRT/TO/FLAG/HA-Dest plasmid
Site-directed mutagenesis to create the plasmids pFRT-FlagHA-DHX36-iso2, pFRT-FlagHA-DHX36-iso1-E335A, and pFRT-FlagHA-DHX36-iso2-E335A

dependent variables

Expression of DHX36 isoforms and mutants in HEK293 T-Rex Flp-In cells

control variables

Full-length complementary DNA of wild-type HEK293 T-Rex Flp-In cells used as template for cloning
2× Phusion PCR Master Mix used for site-directed mutagenesis
PcDNA5-FRT-GFP-mCherry-3pGW backbone used for reporter gene plasmids
BP and LR clonase systems used for generating reporter gene plasmids

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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