chemically synthesized gene fragments  (Twist Bioscience)

Journal: Science Advances

Article Title: Hepatovirus translation requires PDGFA-associated protein 1, an eIF4E-binding protein regulating endoplasmic reticulum stress responses

doi: 10.1126/sciadv.adq6342

Figure Lengend Snippet: ( A ) Immunoblots of sgCtrl and PDAP1-KO1.4 cell lysates collected 12 hours after tunicamycin (Tm) treatment. ( B ) Dicistronic HAV IRES reporter activity in tunicamycin-treated Huh-7.5 cells. Cells were transfected with plasmid DNA 1 hour after addition of tunicamycin and harvested 24 hours later for FLuc and RLuc assays. N = 3 technical replicates from a representative experiment. P values by one-way ANOVA. LU, light units; AU, arbitrary units. ( C ) NLuc activity reflecting replication of the CHIKV-NLuc reporter virus in sgCtrl and PDAP1-KO1.4 cells 24 hours after infection. MOI, multiplicity of infection. ( D ) Immunoblots of CHIKV capsid protein, p-eIF2a, and eIF2a in lysates from sgCtrl and PDAP1-KO1.4 cells 24 hours after CHIKV-NLuc infection. ( E ) Dicistronic HAV IRES reporter [see (B)] activity in CHIKV-NLuc–infected sgCtrl cells. Cells were transfected with plasmid DNA 1 hour after infection and harvested 24 hours later for FLuc and RLuc assays. P values by one-way ANOVA. Data shown represent N = 3 technical replicates. ( F ) Immunoblots of GFP expressed by circRNA IRES reporters containing BiP, c-Myc, and XIAP IRES sequences in tunicamycin-treated sgCtrl versus PDAP1-KO1.4 cells. ( G ) BiP, c-Myc, and XIAP translational efficiencies in sgCtrl and PDAP1-KO1.4 cells, with and without tunicamycin treatment, based on GFP expression normalized to circRNA abundance measured by RT-qPCR. P values by two-way ANOVA, with corrections for multiple comparisons using the Benjamini, Krieger, and Yekutieli method. N = 3 independent experiments.

Article Snippet: Infectious molecular HAV clones pHAV/p16.2 , pHAV/18f.2 , p18f-NLuc and p18f-NLuc/GAA , p18f-FLuc and p18f-FLuc/GAA replicon clones , pRV-B14-NLuc , pPV-1/FLuc ( ) replicons, and bicistronic HAV IRES reporter pFL-HAV-RL ( ) have been described previously. pPDAP1-Flag, encoding C-terminal Myc/Flag-tagged human PDAP1 (NM_014891.7) under the control of the cytomegalovirus promoter, was purchased from Origene (pRC20058). pPDAP1-Y124 was generated from pPDAP1-Flag by PCR mutagenesis. pMCSG9-PDAP1-Avi, a bacterial expression vector encoding maltose-binding protein fused to PDAP1 with an intervening tobacco etch virus cleavage sequence, was constructed by inserting the PDAP1 sequence from pPDAP1-Flag into pMCSG9 ( ); a C-terminal GLNDIFEAQKIEWHE Avi tag was added by PCR-based mutagenesis. circRNA reporter plasmids for the EMCV (TR-circGFP), KSHV vFLIP, and PV IRES have been described previously ( , ). circRNA reporter plasmids containing IRES elements existing in transcripts encoding BiP (HSPA5, GRP78) (NM_005347.5, nucleotides 1 to 220) , XIAP (NG_007264.1, nucleotides 30293 to 30468) , and c-Myc (NM_001354870.1, nucleotides 798 to 1205) ( ) IRES elements were constructed by Gibson assembly using chemically synthesized DNA gene fragments (Genewiz, Azenta).

Techniques: Western Blot, Activity Assay, Transfection, Plasmid Preparation, Virus, Infection, Expressing, Quantitative RT-PCR