ADAR1 Responsive Luciferase Reporter HEK293 Cell Line
ADAR1 Responsive Luciferase Reporter HEK293 Cell Line is a HEK293 cell line designed to respond to ADAR1 enzymatic activity. These cells were engineered to express an ADAR1 reporter construct comprised of an ADAR1 hairpin target with a stop codon (UAG) susceptible to ADAR1-mediated editing to tryptophan (UUG), located upstream of a firefly luciferase reporter (Figure 1).
This cell line has been validated by comparing reporter activation after transfection with ADAR1 and ADAR2. This cell line has been used for the development of the constitutive ADAR1 expressing ADAR1 Activity Luciferase Reporter HEK293 Cell Line (#82239).
Figure 1: Illustration of the mechanism of action of ADAR1 Responsive Luciferase Reporter HEK293 Cell Line.
The ADAR1 reporter construct is compromised of an ADAR1 hairpin targer with a stop codon (UAG) upstream of the sequence encoding luciferase. In the presence of ADAR1 activity, as in the case of transfection with ADAR1, adenine is converted into inosine encoding now the amino acid tryptophan (UUG) and enabling transcription and expression of luciferase. In the absence of transfected ADAR1, luciferase is not transcribed, and the cells show background luciferase activity. Luciferase activity directly correlated with ADAR1 activity.
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Materials Required for Cell Culture
| Name | Ordering Information |
| Thaw Medium 1 | BPS Bioscience #60187 |
| Growth Medium 1N | BPS Bioscience #79801 |
Materials Required for Cellular Assays
| Name | Ordering Information |
| ADAR1 Activity Luciferase Reporter HEK293 Cell Line | BPS Bioscience #82239 |
| Plasmids encoding ADAR1 isoforms and ADAR2 | |
| Lipofectamine™ 2000 Transfection Reagent | Thermo Fisher #11668027 |
| Assay Media: Assay Medium 1A | BPS Bioscience #79805 |
| Thaw Medium 1 | BPS Bioscience #60187 |
| 96-well tissue culture white, clear-bottom assay plate | Corning #3610 |
| ONE-Step™ Luciferase Assay System | BPS Bioscience #60690 |
| Luminometer |
The cell line has been screened to confirm the absence of Mycoplasma species.
ADAR (Adenosine Deaminase Acting on RNA) enzymes perform adenosine to inosine base editing in RNA, particularly targeting adenosines located within a specific double-stranded stem-loop motif (Figure 1). In the context of healthy, uninfected cells, ADAR1 performs A-to-I editing on endogenous double-stranded RNA to prevent it from activating the downstream dsRNA sensors RIG-I (retinoic acid-inducible gene I) and MDA5 (melanoma differentiation-associated protein 5), which in-turn activate a pro-inflammatory response. Loss of function mutations in ADAR1 result in aberrant activation of the dsRNA sensors and are involved in autoimmune disorders. ADAR1 dysfunction also impacts cancer cell growth, proliferation, and response to immunotherapy. ADAR1 expression is increased in many tumor types and ADAR1 knock-out has been demonstrated to improve the response to certain immunotherapies, such PD-1 (programmed death protein 1)/PD-L1 (programmed death ligand 1) blockade, and to circumvent tumor immunotherapy resistance mechanisms, making ADAR1 an attractive target for therapeutic development.
HEK293 cells express low levels of endogenous ADAR1, therefore this cell line is ideal for studies of genetically engineering variants of ADAR1. For example, one can compare the activity of ADAR1 variants introduced into the cells by transfection or transduction. The use of luciferase as reporter allows for an easy assay read-out, making this cell line an attractive cellular system for ADAR1 studies.
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