High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

Summary

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

Abstract

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson's disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

Introduction

The ability to identify key genetic regulatory elements and the factors that act on them is fundamental for the exploration of numerous biological processes. However, identifying factors that regulate expression of genes in rare cell types, such as specific neuronal populations, can be challenging. Here, we present a protocol for identifying novel transcriptional regulators of alpha-synuclein (SNCA), a gene associated with Parkinson's disease and expressed in dopaminergic neurons in the substantianigra pars compacta region of the midbrain. We accomplish this using a high-throughput, dual-luciferase, in vitro reporter screen to deconstruct alpha-synuclein expression in 293T cells. The alpha-synuclein promoter is first cloned into a reporter plasmid containing the firefly luciferase gene. A commercially available plasmid containing the Renilla luciferase under the control of a constitutively active promoter serves as an internal control. These reporter constructs are co-transfected into 293T cells in microplates with plasmids from a DNA expression library, such that each well is transfected with a single library plasmid. After 48 hr, luciferase activity for each reporter is measured sequentially using a dual-glow assay. The relative expression of alpha-synuclein in response to each library gene is inferred by the ratio of firefly:Renilla luciferase activity in each well (F:R ratio) after plate-based normalization.

This protocol provides the ability to screen a large number of genes (~2,500 per week) for their ability to transactivate a reporter gene using minimal manpower (1-2 people) and minimal cost (about $3 reagent cost per microplate assay). Transcriptional regulators of neuronal genes (e.g., alpha-synuclein), which are difficult to study in cultured neurons refractory to genetic manipulation, can be compared side-by-side in this direct functional assay. We include in our protocol a detailed method for cloning and growth of plasmids containing the alpha-synuclein promoter, since we found that plasmids containing this region are unstable when grown with conventional procedures (see Discussion). We also include an inexpensive alternative to commercially available dual-luciferase reagents for high-throughput assays. This protocol can be easily adapted to target other regulatory elements of interest, or to any process requiring high-throughput transient transfections.

Protocol

For an experimental overview, see Figure 1.

1. Prepare Reporter Plasmids Containing the Alpha-Synuclein Promoter

Regulatory elements of the alpha-synuclein gene (Figure 2) span approximately 10 kb, from the upstream NACP (non-A beta component of amyloid peptide) dinucleotide repeat sequence^1,2 through intron 2 ³. We include a portion of this region in our reporter construct. We found that plasmids containing intron 2 required special procedures for growth, as outlined below. The luciferase plasmids, pGL4.10 and pGL4.75 (Figure 3), are commercially available from Promega and can be propagated using conventional molecular biology protocols.

1. Amplify the 2 components of the alpha-synuclein promoter in separate PCRs using the recipe in Table 1 and the primers in Table 2.
2. Verify PCR products on an agarose gel and clean using the Qiaquick PCR Purification kit (Qiagen), eluting in 50 μl of TE (Tris-EDTA). Store the eluted 0.9 kb fragment at -20 °C for future use.
3. Digest the entire volume of the 3.4 kb PCR fragment, as well as 5 μg of pGL4.10, with SacI and XhoI. Treat the vector with Calf Intestine Alkaline Phosphatase (Roche) and gel purify using the PureLink Quick Gel Extraction kit (Invitrogen).
4. Ligate the fragment and vector using T4 DNA ligase (NEB). Clean the completed reaction using the PureLink PCR Micro kit (Invitrogen), eluting in 10 μl TE buffer.
5. Transform 2 μl of the cleaned, ligation reaction into 20 μl of ElectroMAX Stbl4 competent cells (Invitrogen) and electroporate according to the manufacturer's instructions. Shake in a 30 °C incubator at 225 rpm for 90 min. Spread 2 volumes on LB plates containing 100 mg/ml ampicillin and incubate at 30 °C for 2 days.
6. Using a toothpick, select 4-6 small colonies for inoculation into 2 ml of TB (Terrific Broth). Grow these miniprep cultures in a 30 °C shaker O/N.
7. Purify plasmid DNA from 1.5 ml of each miniprep culture using the PureLink HiPure Plasmid Miniprep kit (Invitrogen).
8. Digest the minipreps with BamHI and electrophorese on an agarose gel. The correct clone should produce fragments of 2.8 kb and 4.9 kb.
9. Restreak the remaining miniprep culture from the correct clone on a fresh LB (Luria Broth) plate and grow at 30 °C for 2 days.
10. Select a small colony and inoculate a 1.5 ml TB starter culture. Shake O/N at 30 °C. Do not let the starter culture grow to saturation.
11. Dilute the entire starter culture into 150 ml of prewarmed TB and shake at 30 °C for 2 days. Before proceeding to the next step, use 1.5 ml of this culture for an additional miniprep and digestion to confirm that the clone did not mutate during replication.
12. Purify plasmid DNA from the remaining culture using the PureLink HiPure Plasmid Maxiprep kit (Invitrogen). Expected yields of this intermediate plasmid are 50-150 μg per 150 ml of culture.
13. Thaw the 0.9 kb fragment frozen in Step 1.2. Repeat Steps 1.3 to 1.12 to clone the 0.9 kb fragment into the intermediate plasmid, digesting instead with KpnI and SacI. Ligate, transform, select, and grow for maxipreps as above. Digestion with BamHI should yield fragments of 5.8 kb and 2.8 kb. This is the plasmid that will be used for the screen.

2. Prepare 293T Cells, Reporter Plasmids, and Library DNA for Screening

293T cells are first seeded into 96-well plates and transfected the following day. Reporter plasmids and library DNAs are diluted into 96-well plates. We obtained plates of transfection-grade library DNA from the DNA Core at Massachusetts General Hospital (dnacore.mgh.harvard.edu). This protocol transfects a single library plate into 2 identical plates of 293T cells (Figure 1), thus 2 plates of cells should be seeded for each library plate to be screened.

Note: Grow cells in media without phenol red or antibiotics, as these interfere with the luciferase assays and increase toxicity during transfection, respectively.

1. For each library plate to be screened, resuspend trypsinized 293T cells to a concentration of 1.5 x 10⁵ cells/ml in DMEM containing 14% FBS. Pour into a sterile reservoir (Axygen).
2. Place the reservoir, 2 clear-bottomed, 96-well plates (Corning), and a box of filter pipet tips (Agilent) on the robot deck. Dispense 100 μl of cells into each well of both plates, for a density of 1.5 x 10⁴ cells per well.
3. Centrifuge the cell plates at 50 x g for 2 min, using low ramp speeds to ensure equal cell density throughout each well. Incubate at 37 °C and 10% CO₂ O/N.
4. Dilute the firefly and Renilla reporter plasmids to 5 ng/μl in serum-free media. Combine the dilutions in a ratio of 5:3 firefly to Renilla plasmid (by volume) in a 15 ml conical tube.
5. Pipet the combined plasmids into each well of a 96-well plate, dispensing 17 μl per library plate, plus 2-10 μl excess, into each well.
6. Obtain transfection-grade DNA library plates as above and dilute to 13.3 ng/μl with endotoxin-free water. The minimum volume per well should be 28 μl.

3. Perform Transfections

On day 2 of the screen, reporter plasmids and library DNAs are transfected into 293T cells.

1. For each library plate, mix 107.5 μl of RT Optifect (Invitrogen) with 4.3 ml serum-free media (1:40 dilution) in a polystyrene tube. Incubate for 5 min at RT, and then dispense 44 μl (per library plate) into each well of a 96-well, polystyrene V-bottomed plate (Greiner).
2. Place the plate of diluted Optifect, reporter plasmids (Step 2.5), library DNAs (Step 2.6), and a box of pipet tips on the robot deck.
3. Aspirate 17 μl of reporter plasmids, 43 μl of diluted Optifect, and 26 μl of library DNA, inserting a 5 μl air gap between each aspiration. The library DNA should be aspirated last to prevent cross-contamination. Dispense the contents of the tips into a new polystyrene V-bottomed plate, mix, cover, and set aside for 20 min to allow lipid/DNA complexes to form. If transfecting multiple library plates, replace the pipet tips and library plate, and repeat.
4. Uncover the Optifect/DNA mixture plate and place it on the robot deck with 2 plates of 293T cells. Using a single box of pipet tips, aspirate 80 μl of the Optifect:DNA mixture and slowly dispense 40 μl onto each plate of cells. If transfecting multiple library plates, repeat for all Optifect:DNA plates. Cover cells.
5. Centrifuge the cell plates at 1,200 x g for 30 min. Cover and return to the incubator.
6. Incubate the cells for 4-6 hr. During this incubation, prepare fresh media by mixing 12 ml DMEM containing 10% FBS and 10 μg/ml ciprofloxacin (CellGro) for each transfected library plate. Pour fresh media into a sterile reservoir.
7. Place 2 boxes of robot tips, a single plate of cells, the reservoir containing fresh media, and a waste reservoir onto the robot deck. Aspirate 100 μl of media from the cells and dispense into the waste reservoir partially filled with 70% ethanol. Using fresh tips, aspirate 100 μl of fresh media and slowly dispense onto the cells. Repeat for all plates of cells.
8. Return plates to the incubator for 48 hr.

4. Perform Dual-Luciferase Assays

On day 4 of the screen, the firefly and Renilla luciferase assays are performed.

Note: The dual-luciferase assay requires the sequential addition of 2 assay buffers, 3X firefly assay buffer and 3X Renilla assay buffer, as described in Table 3. Firefly and Renilla luciferases are not secreted, thus cells must be lysed prior to measuring luciferase activity. Firefly assay buffer lyses cells and provides substrate for the firefly luciferase; Renilla assay buffer quenches the firefly signal and provides substrate for the Renilla luciferase.

1. For each library plate, prepare 8 ml of 3X firefly assay buffer from stock solutions as described in Table 3, and dispense 82 μl into each well of a 96-well V-bottomed plate.
2. For each library plate, also prepare 12 ml of 3X Renilla assay buffer and dispense 122 μl into each well of a 96-well V-bottomed plate. Set aside both the firefly and Renilla assay buffers.
3. Place a box of pipet tips, a waste reservoir, and 2 plates of cells (transfected from the same library plate) on the robot deck.
4. Aspirate 60 μl of media from each plate and discard in the waste reservoir partially filled with 70% ethanol. Cover plates and set aside. If transfecting multiple library plates, repeat for all sets of plates.
5. Place 2 boxes of pipet tips, the plate of 3X firefly assay buffer, and a set of cell plates on the robot deck.
6. Aspirate 40 μl of 3X firefly assay buffer and add it to the first plate of cells, mixing thoroughly. Switching to new pipet tips, repeat for the second cell plate. Place cells at RT for 10 min to complete lysis. If transfecting multiple library plates, replace the boxes of tips and cell plates, and repeat.
7. Record luminescence from each well of each cell plate on a luminometer, recording for 1 sec per well. Return plates to the robot deck.
8. Place 2 boxes of pipet tips, the plate of 3X Renilla assay buffer, and a set of cell plates on the robot deck.
9. Aspirate 60 μl of 3X Renilla assay buffer, and add it to the first plate of cells, mixing thoroughly. Switching to a new box of pipet tips, repeat for the second cell plate. If transfecting multiple library plates, repeat for all sets of cell plates.
10. Record luminescence from all plates on a luminometer, recording each well for 1 sec as above. Discard plates.

5. Data Analysis

1. Calculate the firefly:Renilla luminescence ratio ("F:R ratio") for each well by dividing the counts of firefly signal by the counts of Renilla signal.
2. Calculate the induction value by dividing the F:R ratio of each well by the average F:R ratio of the plate, excluding the highest and lowest 25% of wells.
3. Average the induction values across replicates for a single transfected library plate. Genes inducing or repressing alpha-synuclein more than three-folds are considered "hits" in this screen and are subject to further validation and secondary screens.

Results

Typical luciferase values, F:R ratios and induction values for a single half-plate are shown in Figure 4. Note the hit in well H2, a 32-fold inducer. Genes causing excessive toxicity (for example, well E3), or wells that were poorly transfected, will produce low values for both firefly and Renilla luciferases, but an average induction value. Genes causing non-specific induction of luciferase activity, perhaps via interaction with the pGL4 backbone, will induce firefly and Renilla

Discussion

Alpha-synuclein has been implicated in Parkinson's disease (PD) as a component of Lewy bodies⁴, intracellular inclusions considered pathognomonic for the disease. Numerous genome-wide association studies have linked single nucleotide polymorphisms in alpha-synuclein with increased risk for sporadic PD^5,6,7. Although less common than sporadic PD, familial PD may be also caused by mutations in alpha-synuclein⁸, as well as duplication and triplication of the alpha-synuclein locus^9,1...

Materials

Name	Company	Catalog Number	Comments
Material
QIAQuick PCR Purification Kit	Qiagen	28106
PureLink Quick Gel Extraction Kit	Invitrogen	K2100-12
PureLink PCR Micro Kit	Invitrogen	K310250
PureLinkHiPure Plasmid Miniprep Kit	Invitrogen	K2100-03
PureLinkHiPure Plasmid Maxiprep Kit	Invitrogen	K2100-07
Bravo Robot	Agilent	-
Robot Pipet Tips with Filter	Agilent	19477-022
Robot Pipet Tips without Filter	Agilent	19477-002
Clear-bottomed 96-well Plates	Corning	3610
Reservoirs	Axygen	RES-SW96-HP-SI
Polystyrene V-bottomed 96-well Plate	Greiner	651101
Polypropylene V-bottomed 96-well Plate	Greiner	651201
Adhesive Plate Covers	CryoStuff	#FS100
Reagent
Phusion DNA Polymerase	New England Biolabs	M0530L
BAC 2002-D6	Invitrogen	2002-D6
Calf Intestine Alkaline Phosphatase	Roche	10713023001
T4 DNA Ligase	New England Biolabs	M0202L
pGL4.10	Promega	E6651
pGL4.74	Promega	E6931
ElectroMAX Stbl4 Competent Cells	Invitrogen	11635-018
Subcloning Efficiency DH5α Competent Cells	Invitrogen	18265-017
DMEM without Phenol Red	Invitrogen	31053-028
Optifect	Invitrogen	12579017
Ciprofloxacin	CellGro	61-277-RF
Tris-Hydrochloride Powder	Sigma	93287
Tris-Base Powder	Sigma	93286
Triton X-100 Pure Liquid	Fisher	BP151-100
DTT	Invitrogen	15508-013
C–nzyme A	Nanolight	309
ATP	Sigma	A6419
Luciferin	Invitrogen	L2912
h-CTZ	Nanolight	301
PTC124	SelleckBiochemicals	S6003