Reporter gene assays are widely used to study the regulation of gene expression. This has only previously been possible using exogenous genetic overexpression constructs containing the regulatory element of interest, sequences required for the formation of functional mRNA and the reporter gene sequence.
Horizon Discovery has developed an innovative advance in reporter technology by combining there gene editing platform with Promega’s NanoLuc® luciferase. A series of NanoLuc®-PEST promoter reporter cell lines have been generated to introduce NanoLuc® luciferase under the control of endogenous promoters of genes of interest. NanoLuc® luciferase is a small monomeric enzyme (19.1kDa) engineered for optimal performance as a luminescent reporter. The enzyme is about 150-fold brighter than either firefly (Photinus pyralis) or Renilla reniformis luciferase and permits evaluation of gene expression without the need for overexpression. The engineered cell lines have a variety of baseline luciferase signals giving a true reflection of the different transcription levels of the genes of interest.
NanoLuc-PEST promoter reporter cell lines enable the evaluation of the regulation of endogenous gene expression in a rapid, simple assay system, suitable for use on higher throughput platforms. The use of reporters that enable evaluation of endogenous gene expression rather than relying on overexpression approaches represents a significant advance for understanding and screening true biological responses.
Below we detail two examples of gene transciption modulation that can be monitored at the endogenous level using these reporter lines.
Actinomycin D is a non-specific inhibitor of gene transcription. Treatment of the HIF1A, MYC and GLI1 NanoLuc®-PEST promoter reporter cell lines with actinomycin D resulted in a dose dependent decrease in signal (Figure 1). Data obtained from multiple independent assays was extremely robust and reproducible. IC50s generated were similar between cell lines, as expected for a non-specific inhibitor. The assay was multiplexed with CellTiter-Blue® to determine cellular viability and demonstrated that changes in signal were due to effects on transcription only.
Figure 1. Treatment with actinomycin D results in a dose dependent decrease in luciferase signal. NanoLuc®-PEST promoter reporter cell lines were treated with actinomycin D for 6h. NanoLuc® luciferase signal was measured to determine levels of transcription and the assay was multiplexed with CellTiter-Blue® as a measure of viability. Multiple repeats were performed and data shown is a combination of at least three independent assays.
Actinomycin D inhibits transcription by intercalating into DNA1 and at low doses induces a DNA damage response (DDR). This was demonstrated by the response of the P21 NanoLuc-PEST promoter reporter cell line to actinomycin D treatment, since p21 is a member of the DDR pathway2. An increase in luciferase signal was seen at low concentrations of actinomycin D, reflecting an increase in p21 transcription, followed by inhibition of transcription at higher concentrations (Figure 2A). An increase in expression of p21 protein and other markers of DNA damage was confirmed by Western blotting (Figure 2B). Critically, the response of the unmodified parental cell line was comparable to the reporter cell line, highlighting the applicability of these reporters for monitoring modulation of biological effects.
Figure 2. Treatment of the P21 NanoLuc®-PEST promoter reporter cell line with actinomycin D induces a DDR at low doses. The P21 NanoLuc®-PEST promoter reporter cell line was treated with actinomycin D for 6h. (A) P21 transcription was measured using Nano-Glo® luciferase and the assays were multiplexed with CellTiter-Blue® to give a measure of cell viability. (B) Western blotting confirmed the induction of a DDR, with results consistent between the unmodified parental and P21 NanoLuc®-PEST promoter reporter cell lines. Abbreviation: c, vehicle control.
The P21 NanoLuc-PEST promoter reporter cell line was treated with DNA damaging agents. Treatment with both etoposide and doxorubicin led to dose dependent DDRs. P21 transcription was increased as demonstrated by an increase in luciferase signal in the promoter reporter cell line (Figure 3A). Western blotting confirmed an increase in p21 protein levels and expression of other markers of DDR (Figure 3).
Figure 3. Etoposide and doxorubicin induce p21 transcription as part of a DDR. The P21 NanoLuc-PEST promoter reporter cell line was treated with etoposide and doxorubicin for 6h, to induce a DDR. (A) P21 transcription was measured using Nano-Glo® luciferase and the assays were multiplexed with CellTiter-Blue® to give a measure of cell viability. (B) Western blotting confirmed the induction of a DDR, with results consistent between the unmodified parental and P21 NanoLuc-PEST promoter reporter cell lines