The dAAVS1–TetBxb1BFP plasmid was created by using the plasmid backbone and homology arms from AAV–CAGGS–EGFP (Addgene #22212), rtTA3G transactivator from pLenti–CMV–rtTA3-Blast (Addgene #26429), mTagBFP2 sequence from mTagBFP2–Actin-7 (Addgene #55273), and the Tet-responsive promoter from pLVX–TRE3G–mCherry (Clontech). Deletion of internal sequences from dAAVS1–TetBxb1BFP was used to create the dAAVS1–rtTA3G and dAAVS1-Dummy plasmids. The dAAVS1–TetBxb1BFP–BC plasmids were created by adding 15 degenerate nucleotides between the Tet-responsive promoter and the upstream BGH terminator. AAVS1 genomic disruption to generate HEK 293T candidate landing pad clones was performed either with TALENs (Addgene #59025 and #59026) or pSpCas9-2A-GFP (Addgene #48138) and AAVS1 gRNA T2 (Addgene #41818). The Bxb1 expression vector pCAG–NLS–HA–Bxb1 (Addgene #51271) was used to express Bxb1 and stimulate recombination. The attB-mCherry, attB-EGFP, attB-Ub3kGiM and attB-3kGiM recombination plasmids were created using coding sequences from p2attPC (Addgene #51547), pIRES2-DsRed-Express (Clontech), and AAV–CAGGS–EGFP. Other recombinant DNA elements were generated by oligonucleotide synthesis (Integrated DNA Technologies). All molecular cloning steps were performed with Gibson assembly (20 (link)). All primer and plasmid sequences can be found in Supplementary Table S2 and Supplemental Text 1 .
Plvx tre3g mcherry
Manufactured by Takara Bio
Sourced in United States
The PLVX-TRE3G-mCherry is a lentiviral vector that allows for the inducible expression of the mCherry fluorescent protein. The vector contains a Tet-On 3G inducible promoter system, which enables tight control of gene expression in response to the presence of doxycycline.
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Lab products found in correlation
Pcag nls ha bxb1, by Addgene (1 mentions)
Plenti cmv rtta3 blast, by Addgene (1 mentions)
Pires2 dsred express, by Takara Bio (1 mentions)
In fusion, by Takara Bio (1 mentions)
Ptre3g mcherry, by Takara Bio (1 mentions)
Pcmv tet3g, by Takara Bio (1 mentions)
Geneclean kit, by MP Biomedicals (1 mentions)
Tet on 3g inducible expression system, by Takara Bio (1 mentions)
3 protocols using plvx tre3g mcherry
1
Modular Platform for Inducible Gene Expression
2
Engineered Expression Vectors for Protein Research
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Expression vectors for stable overexpression of the proteins Clover-PPARγ, roGFP2, and Grx1-roGFP2 were generated by replacing EGFP of pHR′SIN-cPPT-SE [22 (link)] by the appropriate coding sequences due to In-Fusion® (Clontech, Takara, Japan) recombination as previously described [23 (link)]. In brief, the plasmids pcDNA3-Clover [24 (link)], pDsRed-Monomer-C1-hPPARγ1 [25 (link)], and pQE-60_Grx1-roGFP2 [26 (link)] were used as templates. N- and C-terminally HA-tagged hPPARγ was generated by the same approach, elongated by an IRES sequence derived from the pLVX-TRE3G-mCherry (Clontech), and an additional Clover-sequence to generate a hPPARγ-IRES-Clover expression construct. Therefore, hPPARγ was fused to the HA-coding sequence 5′-ATGTACCCATACGATGTTCCAGATTACGCT -3′ at the N-terminus and to 5′-CCCCCTCCGCCCCCACCTTACCCATACGATGTTCCAGATTACGCT TGA-3′ at the C-terminus. Cysteine to alanine mutants of hPPARγ were created by mutagenesis of the TGX into GCX codons using PfuII polymerase (Agilent Technologies Deutschland GmbH, Böblingen, Germany). The same mutagenesis approach was used for the generation of serine to alanine (TCT to GCT) and serine to glutamic acid (TCT to GAG) mutants of hPPARγ.
3
Inducible Expression of ldrB Gene
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Tet-On® 3G Inducible Expression System containing pTRE3G-mCherry and pCMV-Tet3G vectors was purchased from Clontech. The ldrB gene flanked by the NdeI site ant 5′ and EcoRI site at 3′ was synthesized and subcloned into pLVX-TRE3G-mCherry (Clontech, Palo Alto, CA, USA) previously digested with the same restriction enzymes. The construct was subjected to electrophoresis in 1% agarose gel. Subsequently, the band used was purified using the Gene-clean Kit (MP Biomedicals, Irvine, CA, USA). The vector and the insert were subjected to a ligation reaction of cohesive ends, which allowed proper orientation of the insert. Then, to verify the ligation between the vector and the ldrB gene, on one hand, a complete sequencing of the vector was performed, and, on the other hand, the construction was digested with XhoI and ApaI restriction enzymes, and an agarose gel electrophoresis was performed. The appearance of two bands corresponding to the 1187 pb and 8 kb confirmed the correct structure of our construction (Figure 1 A). This construction was used to transform competent E. coli bacteria, which allowed us to obtain the quantities necessary for subsequent transfection experiments.
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