pYSYE0002 is a prey plasmid containing the DAF-12 N500 gene expressed from the copper inducible cup-1 promoter and pYSYR0002 is a bait plasmid contain a DAF-12 responsive element cloned upstream of Pcyc-1 driving lacZ expression [40].
A boundary element cloned between the locus control region (LCR) enhancer and the γ-neo gene providing antibiotic resistance blocks promoter activation, thereby rendering the cells sensitive to G418 and decreasing the number of colonies obtained in this assay.
MVLN cells (generously provided by Dr M Pons, INSERM U58, Monpellier, France) are MCF-7 cells stably transfected with the oestrogen-response element cloned upstream the luciferase reporter gene (Demirpence et al, 1993).
In order to assess whether NF-κB and AP-1 cooperation was required for the up-regulation of IL-8 mRNA levels in HCT-15 cells, we used the IL-8 promoter element cloned into a luciferase reporter construct with wild type or mutated NF-κB and AP-1 binding sites.
We used the QuikChange Lightning site-directed mutagenesis kit from Agilent Technologies (Santa Clara, CA) and the ISX element cloned in Ellison and Bachtrog (2013) to engineer four ISX variants that differed only with respect to the TT haplotype: ISX-GA, ISX-GT, ISX-TA, and ISX-TT.
"This is nuclear transfer, one element of cloning.
To minimize interference between the different tetracycline-inducible promoters used in this study (tetracycline-responsive element (TRE), TRE-tight, or Tk-tetO) and the rtTA-M2 transactivator, both elements were cloned in opposite directions and separated by a 5 kb human p53 intron.
Potential regulatory elements were cloned into pGL2-Promoter (Promega) and transfected into MCF7 cells using the TransIT-LT1 Transfection Reagent (Mirus).
Separate enhancer elements are cloned upstream of an hsp70 promoter.
All elements were cloned into reporter vectors and assessed for expression modulation in dual luciferase reporter assays.
