gateway lr reaction mixing destination p5e ubi loxtagbfp  (Addgene inc)

Journal: Nucleic Acids Research

Article Title: Cre-Lox miRNA-delivery technology optimized for inducible microRNA and gene-silencing studies in zebrafish

doi: 10.1093/nar/gkaf004

Figure Lengend Snippet: Schematics of the new RNAi backbone for zebrafish gene silencing. ( A ) Tol2kit compatible p3E presenting an optimized empty synthetic pri-miR (or RNAi cassette) embedded into a β-globin intronic sequence. This presented pri-miR is designed to allow rapid directional insertion of synthetic pre-miR of choice. Synthetic p re-miR(s) of choice are generated by annealing specific top and bottom RNAi oligos designed to release a mature miRNA directed against the 3′UTR of a gene-of-interest [see ‘Methods’ section and previous material ]. The RNAi cassette is flanked by restriction sites allowing subsequent and repetitive chaining [see ‘Methods’ and ) for generating p3E-RNAi plasmid with multiple pri-miR for either increasing the potency of the desired knockdown or targeting multiple genes at the same time. ( B ) Without intron, the pre-miR /RNAi cassette is transcribed along with a co-marker on the same RNA, which is cut by Drosha in the nucleus for releasing the associated pre-miR . This cut leaves the mRNA without polyA tail and leads to its rapid degradation. The associated fluorescence is not a good indicator of the activity and amount of synthetic miRNA produced. ( C ) The presence of the intronic sequence is designed to rescue the co-expression of the marker (see also ).

Article Snippet: The PCR product was further digested using BamHI along with Tol2kit 101_p5E-Ubiquitin plasmid (Addgene #27320) ( ).

Techniques: Sequencing, Generated, Plasmid Preparation, Knockdown, Marker, Fluorescence, Activity Assay, Produced, Expressing

Journal: bioRxiv

Article Title: Multisite Assembly of Gateway Induced Clones (MAGIC): a flexible cloning toolbox with diverse applications in vertebrate model systems

doi: 10.1101/2024.07.13.603267

Figure Lengend Snippet:

Article Snippet: The resulting amplicon was purified by PCR Cleanup (Qiagen) then recombined with pDONR P4-P1r in a BP reaction to generate p5E Glast (JDW 1182). p5E CAG was created by inserting the CAGGS promoter into p5E-MCS via KpnI to BamHI. p5E TetO(8x) CMV minpro was created by subcloning the TRE and downstream minimal CMV promoter from pB-TA-ERN into p5E MCS via PspXI and SacII using standard T4 DNA ligation. p5E-TRE3GV (JDW 1163) was subcloned by digesting JDW 447 (pBT346.6-TRE3G-FRT) with XhoI and HindIII and shuttled into p5E-MCS (JDW 459). p5E Ubi-pro, which contains the zebrafish Ubiquitin promoter, exon1, and intron 1, was made by digesting p5E- Ubi -loxP-EGFP-loxP (Addgene #27322) with BamHI, and ligating the digested product together to remove the loxP flanked EGFP cassette and reconstitute the BamHI site.

Techniques: