ef1a sequence excluding intron pkg1367 ppv1 hpgk addgene  (Addgene inc)

Journal: Journal of Experimental Botany

Article Title: The transcription factor OsWRKY10 inhibits phosphate uptake via suppressing OsPHT1;2 expression under phosphate-replete conditions in rice

doi: 10.1093/jxb/erac456

Figure Lengend Snippet: Expression of the fusion of WRKY10 and Virus Protein 16 (VP16) leads to increased P accumulation. Rice seeds were germinated in sterilized water and supplied with 1/2 strength Kimura B solution until the third leaf blades were fully expanded, and then treated with HP (90 μM) and LP (1 μM) until the sixth leaf blades were fully expanded; four biological repeats are set for each treatment. (A) Phenotype of wild-type (WT) and WRKY10-VP16 transgenic plants grown under HP (left) and LP (right) conditions. Scale bars=10 cm. (B) Phenotype of fourth leaf blades of WT and WRKY10-VP16 under HP (left) and LP (right) conditions. (C) Total P concentration in shoot and root under HP (left) and LP (right) conditions. All data are plotted with box–whisker plots: the whiskers represent maximum and minimum values, and boxes represent the upper quartile, median, and lower quartile. The results shown are from four biological replicates. Data significantly different from the corresponding controls are indicated (* P <0.05, ** P <0.01; Student’s t -test). NC, negative control.

Article Snippet: For transcriptional activator vectors, the VP16 sequence was synthesized by Genscript according to the methods described by Kong et al . (2016) , and was then cloned into a WRKY10 overexpression vector using the ClonExpress II One Step Cloning Kit (Vazyme).

Techniques: Expressing, Virus, Transgenic Assay, Concentration Assay, Whisker Assay, Negative Control

Journal: Journal of Experimental Botany

Article Title: The transcription factor OsWRKY10 inhibits phosphate uptake via suppressing OsPHT1;2 expression under phosphate-replete conditions in rice

doi: 10.1093/jxb/erac456

Figure Lengend Snippet: Alteration in the expression of three PHT1 genes in wrky10 mutants and WRKY10-VP16 plants. Rice seeds were geminated in sterilized water and supplied with 1/2 strength Kimura B solution until the third leaf blades were fully expanded, and then treated under +P (90 μM) and –P (0 μM) conditions until the sixth leaf blades were fully expanded; the root was harvested for RNA extraction and RT–qPCR. PHT1 gene expression was detected in wrky10 mutants (A) and WRKY10-VP16 plants (B). All data are plotted with box–whisker plots: the whiskers represent maximum and minimum values, and boxes represent the upper quartile, median, and lower quartile. The results shown are from four biological replicates. Data significantly different from the corresponding controls are indicated (* P <0.05, ** P <0.01; Student’s t -test).

Article Snippet: For transcriptional activator vectors, the VP16 sequence was synthesized by Genscript according to the methods described by Kong et al . (2016) , and was then cloned into a WRKY10 overexpression vector using the ClonExpress II One Step Cloning Kit (Vazyme).

Techniques: Expressing, RNA Extraction, Quantitative RT-PCR, Gene Expression, Whisker Assay

Journal: ACS Synthetic Biology

Article Title: Enhanced MyoD-Induced Transdifferentiation to a Myogenic Lineage by Fusion to a Potent Transactivation Domain

doi: 10.1021/sb500322u

Figure Lengend Snippet: MyoD lentiviral delivery system. WT MyoD, VP64 MyoD, VP16 MyoD, and p65 MyoD were cloned into a Tet-ON lentiviral vector. This vector constitutively expresses the reverse tetracycline transactivator (rtTA2 S -M2) and the puromycin resistance gene (Puro R ) from the human phosphoglycerate kinase (hPGK) promoter. The rtTA2 S -M2 and Puro R are coexpressed from the same mRNA via an internal ribosomal entry site (IRES). The MyoD-T2A-dsRed Express2 expression cassette is downstream of the tetracycline response element (TRE) promoter. The rtTA2 S -M2 binds to the TRE and activates expression of the downstream genes in the presence of doxycycline. The T2A ribosomal skipping peptide results in the expression of two separate proteins from a single mRNA. The peptide sequence of each transcriptional activation domain is shown. The minimal activation domain of VP16 is shown in red. PKKKRKV is the SV40 nuclear localization signal and p65 contains the native nuclear localization signals KRKR and LGALL (underlined). VP64 also contains an HA epitope tag (YPYDVPDYA) that was not utilized in this study (underlined). All fusion proteins contain a flexible serine/glycine linker GGSGGGS (bold) between the activation domain and MyoD.

Article Snippet: The VP16 sequence was isolated from Addgene plasmid 11351.

Techniques: Clone Assay, Plasmid Preparation, Expressing, Sequencing, Activation Assay