A cell-based assay that can be used for HTP screening approaches has to meet specific criteria, such as scalabilty and, robustness. Moreover, it has to provide a simple but quantifiable readout.
The screening assay described here is mimicking proximal aspects of the Nrg1-ERBB4 signalling using a co-culture assay of two different cell populations (Figure 25). The first population represents PC12 cells expressing constitutively active mouse-derived Nrg1-typeI-1a (Figure 26, Figure 27).
The second population represents also PC12 cells but expressing human ERBB4 fused to the N-terminal part of the TEV protease (NTEV) that is linked via a TEV protease cleavage site (tevS, sequence: ENLYFQ’G) to the artificial transcription factor Gal4-VP16 (GV). As ERBB4 is a type I transmembrane receptor, GV is anchored to the membrane as long as no functional TEV protease is present. Activated ERBB4, i.e. Nrg1 ligand-bound and dimerised ERBB4, is auto-phosphorylated at several specific Tyr residues in the C-terminal region that act as binding sites for phospho-adapter molecules, such as the regulatory subunit of the PIK3, PIK3R1, that binds to phosphorylated Tyr residues within YXXM motifs. Therefore, PIK3R1 fused to the C-terminal part of the TEV protease (CTEV), is also expressed in the second cell population. In this assay, activated ERBB4 binds to PIK3R1initiating the formation of TEV protease activity as both TEV protease fragments get into close proximity, which allows the refolding of the inactive fragments into a functional TEV protease. The protease can cleave at the tevS to release the artificial transcription factor GV that in turn translocates to the nucleus. Here, GV induces the transcription of a firefly luciferase reporter gene that is under the control of upstream activating sequences (UAS-Fluc). UAS-Fluc is the third component that has to be introduced into the second cell population to complete the assay. Using a firefly luciferase as readout allows for a linear amplification of an occurred ERBB4/PIK3R1 interaction event. In addition, the luciferase assay is an easy-to-use readout, stable, and very sensitive assay (Figure 22, Figure 28).
5.1.1 Normalisation
For normalisation purposes, the assay system is co-transfected with a Renilla luciferase vector. The Renilla luciferase is constitutively expressed under the control of the Thymidine kinase (TK) promoter. It is assumed that the transfection efficiency can be compared across all wells using the enzymatic activity of the Renilla luciferase. Choosing the correct promoter for the Renilla
luciferase is vital, as some promoters like the CMV or SV40 promoter seem to respond to growth factors, such as the EGFld and other introduced effects like shRNAs (C. Hammer personal communication). In addition, the normalisation vector in the screen is used to detect toxic substances by means of absolute Renilla values.
5.1.2 Concentrations of plasmids
All plasmids in the assay are regularly used in the concentration of 20ng/well.
Titration assays have shown that this concentration is sufficient to yield optimal induction ratios for the Nrg1-induced ERBB4/PIK3R1 assay, which is also in agreement with a previous report using the split TEV technique for ERBB4 assays (Wehr et al, 2008).
5.1.3 Cell numbers per well
Two different cell concentrations have to be considered. First, cell numbers of population 1 have to be high enough to provide a detectable and stable signal, but adjusted to reasonable levels to prevent overcrowding as cells still divide within the 48h duration of the assay. 40,000 PC12 cells/96-well were determined to be optimal. Second, the Nrg1-typeI-expressing cells have to be adjusted. A number of 5,000-10,000 cells/96-well resulted in a stable activation of the assay that allowed both further activation or inhibition.
5.1.4 Cell types tested for the assay
We tested PC12, HEK293, and CHO cells for usability in the screen. PC12 cells performed best in transient assays. HEK293 cells gave unstable results in transient assays and, unfortunately, a triple transgenic HEK293-derived cell line stopped working reliably. CHO cells gave no stable results at all.
5.1.5 Transfection methods
We tested FuGeneHD, Lipofectamine LTX, and Lipofectamine 2000. For PC12 cells, Lipofectamine 2000 was best suitable, as it could well be used for both adherent and solution transfections. For screening purposes, in-solution transfection was used to increase homogeneity of transfected cells in the wells. A special steering device named “bubble paddle reservoir” available within the Hamilton Microlabstar Plus robot was used to further increase homogeneity of the cells.
5.1.6 Workflow of the NRG1-ERBB4 assay
Critical to HTP-screening are the time points applied in a workflow of a given assay. When using in-solution transfection, two time points are critical. First, the components of the assay have to be expressed and correctly localised to
Results
86 the membrane, a process that occurs during the expression phase of the transiently transfected cells after plating. Second, a good time window has to be chosen for the application of the drugs and concomitant addition of the Nrg1-type1-expressing cells. We tested various setups with six hours differences between each time point and found a reasonable measurement window between 12h expression/12h stimulation and 24h expression/36h stimulation. After a total assay duration of 72h, the plates were overgrown and the cells started to die. For screening purposes, working hours also have to be considered. Therefore, a 24h expression/24 stimulation workflow was chosen.
5.1.7 Stability of the luciferase signals
To provide a robust readout the luciferase signal has to be stable over the time needed for the measurement. For HTP screening, the processing of the assay plates should be as fast as possible. Therefore, cells are lysed using an 8-well dispenser head, allowing to dispense the buffer in minimal time. In addition, the buffer used, the Promega passive lysis buffer, is formulated to provide optimal stability for luciferase enzymes. When the substrates are added to the cell lysates, the luciferase reaction starts immediately. The firefly luciferase signals are relatively stable, with 80% stability over the first 5 minutes. The Renilla luciferase signals decrease fast, with a reduction of 50%
within two minutes. To start the measurement, luciferase substrates are dispensed across the plate, followed by orbital mixing and the measurement itself for which the detector head moves again across the plate. The minimum time required for a measurement of one plate in one run in our luciferase reader is 100 seconds for dispensing, 10 seconds for mixing, and 100 seconds for reading. Thus, firefly and Renilla luciferase activity is measured in each well 110 seconds after addition of the substrate. As the timing for each well regarding substrate addition and reading is the same the initial dramatic decrease in Renilla luciferase activity should not have an effect on the readings obtained from the screen (Figure 21).
Figure 21: Kinetics of firefly and Renilla luciferase
Decay of firefly luciferase (Fluc, blue) and Renilla luciferase (Rluc, red) signal over 20 min.
The Fluc signal drops regularly over 20 min, then remaining with about 50% of activity.
The Rluc signal initially drops fast, losing about 50% of its activity within the first 2 min, but remains relatively stable afterwards.
Results
88 5.1.8 Protocol at a glance one 96 well plate
The experiments lead to the following protocol used in the screening assay:
Transfection Protocol 1. Pipet DNA
2. Mix DNA with 1/2 volume Optimem 3. Vortex
4. Incubate 2-5 minutes
5. add LF2000 to other 1/2 volume Optimem 6. Vortex
7. Incubate 2-5 minutes
8. Combine LF2000/Optimem and DNA/Optimem mix 9. Vortex
10. Incubate 30 minutes at RT
11. Add cells (in medium, with same volume as total volume of Optimem) 12. Incubate 2h at 37°C, but no shaking/rocking
13. Plate cells on 96 well plates, 100µl/96-well. No centrifugation
16. Add EGF-like domain (f.c.10 ng/ml; 20 ng/ml added in 100µl) or PC12 Nrg1-typeI cells (in 100µl)
17. Incubate further 24h
18. lyse cells for assay (35 µl PLB/96-well)