recombinant dna reagent prl tk  (Promega)

Journal: eLife

Article Title: BATF relieves hepatic steatosis by inhibiting PD1 and promoting energy metabolism

doi: 10.7554/eLife.88521

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pRL-TK , Beyotime Biotechnology , D2760 , .

Techniques: Mouse Assay, Transfection, Construct, Virus, Isolation, Infection, Recombinant, Cell Culture, Retroviral, Transduction, Plasmid Preparation, Luciferase, Activity Assay, Sequencing, Synthesized, Reverse Transcription, Cloning, Reporter Gene Assay, Software, Injection, Control

Journal: PLoS ONE

Article Title: Identification and Functional Analysis of Variant Haplotypes in the 5′-Flanking Region of Protein Phosphatase 2A-Bδ Gene

doi: 10.1371/journal.pone.0035524

Figure Lengend Snippet: A. Human PPP2R2D 5′-flanking region identified in the NCBI database. The box with shadow represents exon 1. The four variants identified in current study, −1684 A>G (rs11146169), −1610 A>T (rs77523588), −1196 C>T (rs9419202), and −462 G>A (rs7074421), are located in the 5′-flanking region (−1775 nt to +61 nt) of PPP2R2D . The PPP2R2D genomic reference sequence with GeneBank ID no. 55844 was used with the first nucleotide of the RNA transcript as +1 nt. Other numbers represent primer positions for cloning reporter constructs. B. Fragments F1, F2, and F3 were amplified by PCR to make reporter constructs, and their positions and lengths are shown in parentheses. Each fragment with wild-type alleles was cloned into the pGL3-Basic vector. C. Relative luciferase activity of series truncated constructs of 5′-flanking region of PPP2R2D in human L02 cells. Each construct or empty vector was transfected into L02 cells with pRL-TK plasmid as an internal intrinsic control of transfection efficiency. After 24 hours, the luciferase activities from each testing construct and also from the internal control plasmid were measured using Dual-Luciferase Reporter Assay System. RLU indicates relative light units. The relative luciferase activity was normalized to Renilla activity and was relative to pGL3-Basic control (Ctrl), which was set as 1.0 RLU. Compared with the basal activity of Ctrl, the promoter activity of all constructs was increased at statistically significant levels (* P <0.05). Compared with the activity of the F3 construct, all constructs showed significant lower activity ( # P <0.05). The results represent the mean ± SD of three independent experiments.

Article Snippet: Cells were cotransfected with 50 ng of pRL-TK vector DNA (Promega) and 200 ng of either empty pGL3-Basic plasmid (a promoterless control; Promega) or one of several pGL3b-2R2Dp constructs harboring different lengths and allelotypes or haplotypes of the PPP2R2D promoter.

Techniques: Sequencing, Clone Assay, Construct, Amplification, Plasmid Preparation, Luciferase, Activity Assay, Transfection, Reporter Assay

Journal: Orphanet Journal of Rare Diseases

Article Title: Bi-allelic mutation of CTNNB1 causes a severe form of syndromic microphthalmia, persistent foetal vasculature and vitreoretinal dysplasia

doi: 10.1186/s13023-022-02239-3

Figure Lengend Snippet: Localisation, conservation and transcriptional activity of CTNNB1 c.884C>G; p.(Ala295Gly). A Sanger sequencing chromatograms confirming the presence and zygosity of the CTNNB1 c.884C>G; p.(Ala295Gly) variant in proband and parental DNA samples, in comparison to a control chromatogram which does not contain the sequence change. B CTNNB1 (NM_001904) gene (top) and β-catenin protein (bottom) schematics annotated with previously reported dominant disease-causing mutations (as reported by HGMDpro; orange text = missense mutations, green text = splice-altering mutations; blue text = nonsense mutations; purple text = small insertion, deletion or insertion/deletion mutations) and the location of the CTNNB1 c.884C>G; p.(Ala295Gly) homozygous variant identified by this study (indicated by red text). The biallelic missense mutation identified herein resides within the fourth armadillo (ARM) domain of the encoded protein. C Multispecies alignment of β-catenin (human amino acids 239–298) showing 100% conservation across 28 species of the alanine residue at position 295, found to be mutated to Glycine in the proband described in this report. Red asterisk (*) above the alignments indicates the position of human Alanine 295 in the alignment. Amino acid residues that are fully conserved are represented by an asterisk underneath the alignments; conservation between residues with strongly similar properties are represented by a colon (:); conservation between residues with weakly similar properties are represented by a period symbol (.). D Functional assessment of p.(Ala295Gly) mutant β-catenin on transcription. Bar chart detailing TOPflash luciferase assay results of STF cells transiently co-transfected with wildtype (WT) or mutant (p.(Ala295Gly)) β-catenin or empty vector (pDEST40) and renilla luciferase plasmid. Luciferase activity was measure 48 h post-transfection. Each bar indicates the recorded luciferase activity for each construct following normalisation to renilla activity and relative to the measurement recorded for cells transfected with empty vector. Results are from three independent experiments performed in triplicate. Error bars denote standard error of the mean (SEM); Y-axis represents the fold difference relative to the observed empty vector reading

Article Snippet: 90,000 cells /well were transfected with 399 ng of construct DNA plus 1 ng of Renilla luciferase control plasmid (pRL-TK) (Promega, Madison, Wi, USA), using 1.5μL of FuGENE 6 transfection reagent (Promega).

Techniques: Activity Assay, Sequencing, Variant Assay, Comparison, Control, Mutagenesis, Residue, Functional Assay, Luciferase, Transfection, Plasmid Preparation, Construct