Light-Induced GFP Expression in Zebrafish Embryos using the Optogenetic TAEL/C120 System

Summary

Optogenetics is a powerful tool with wide-ranging applications. This protocol demonstrates how to achieve light-inducible gene expression in zebrafish embryos using the blue light-responsive TAEL/C120 system.

Abstract

Inducible gene expression systems are an invaluable tool for studying biological processes. Optogenetic expression systems can provide precise control over gene expression timing, location, and amplitude using light as the inducing agent. In this protocol, an optogenetic expression system is used to achieve light-inducible gene expression in zebrafish embryos. This system relies on an engineered transcription factor called TAEL based on a naturally occurring light-activated transcription factor from the bacterium E. litoralis. When illuminated with blue light, TAEL dimerizes, binds to its cognate regulatory element called C120, and activates transcription. This protocol uses transgenic zebrafish embryos that express the TAEL transcription factor under the control of the ubiquitous ubb promoter. At the same time, the C120 regulatory element drives the expression of a fluorescent reporter gene (GFP). Using a simple LED panel to deliver activating blue light, induction of GFP expression can first be detected after 30 min of illumination and reaches a peak of more than 130-fold induction after 3 h of light treatment. Expression induction can be assessed by quantitative real-time PCR (qRT-PCR) and by fluorescence microscopy. This method is a versatile and easy-to-use approach for optogenetic gene expression.

Introduction

Inducible gene expression systems help control the amount, timing, and location of gene expression. However, achieving exact spatial and temporal control in multicellular organisms has been challenging. Temporal control is most commonly achieved by adding small-molecule compounds¹ or activation of heat shock promoters². Still, both approaches are vulnerable to issues of timing, induction strength, and off-target stress responses. Spatial control is mainly achieved by the use of tissue-specific promoters³, but this approach requires a suitable promoter or regulatory element, which are not always available, and it is not conducive to sub-tissue level induction.

In contrast to such conventional approaches, light-activated optogenetic transcriptional activators have the potential for finer spatial and temporal control of gene expression⁴. The blue light-responsive TAEL/C120 system was developed and optimized for use in zebrafish embryos^5,6. This system is based on an endogenous light-activated transcription factor from the bacterium E. litoralis^7,8. The TAEL/C120 system consists of a transcriptional activator called TAEL that contains a Kal-TA4 transactivation domain, a blue light-responsive LOV (light-oxygen-voltage sensing) domain, and a helix-turn-helix (HTH) DNA-binding domain⁵. When illuminated, the LOV domains undergo a conformational change that allows two TAEL molecules to dimerize, bind to a TAEL-responsive C120 promoter, and initiate transcription of a downstream gene of interest^5,8. The TAEL/C120 system exhibits rapid and robust induction with minimal toxicity, and it can be activated by several different light delivery modalities. Recently, improvements to the TAEL/C120 system were made by adding a nuclear localization signal to TAEL (TAEL-N) and by coupling the C120 regulatory element to a cFos basal promoter (C120F) (Figure 1A). These modifications improved induction levels by more than 15-fold⁶.

In this protocol, a simple LED panel is used to activate the TAEL/C120 system and induce the ubiquitous expression of a reporter gene, GFP. Expression induction can be monitored qualitatively by observing fluorescence intensity or quantitatively by measuring transcript levels using quantitative real-time PCR (qRT-PCR). This protocol will demonstrate the TAEL/C120 system as a versatile, easy-to-use tool that enables robust regulation of gene expression in vivo.

Protocol

This study was performed with the approval of the Institutional Animal Care and Use Committee (IACUC) of the University of California Merced.

1. Zebrafish crossing and embryo collection

1. Maintain separate transgenic zebrafish lines containing either the TAEL transcriptional activator or the C120-controlled reporter gene to minimize spurious activation.
2. Cross 6-8 adult zebrafish from each line using standard methods⁹ to produce double transgenic embryos that contain both the TAEL and C120 components (Figure 1B).
   NOTE: Alternatively, both components can be expressed transiently through microinjection of mRNA or plasmid DNA using standard methods¹⁰.
3. Collect embryos in Petri dishes containing egg water (60 µg/mL Instant Ocean sea salt dissolved in distilled water), with approximately 30 embryos per condition to be tested.
4. Place the dishes in a lightproof box or cover with aluminum foil to minimize unintended activation by ambient light (see Table 1).

2. Global light induction

1. Use a blue-light (465 nm) LED panel to deliver activating blue light to several embryos at once.
2. Position the LED panel relative to the Petri dishes containing embryos so that the actual power of light received by the embryos is approximately 1.5 mW/cm² as measured by a light power and energy meter (Figure 2A).
3. Pulse the light at intervals of 1 h on/1 h off using a timer relay if illuminating for more than 3 h to reduce the risk of photodamage to the TAEL transcriptional activator^5,8.
   NOTE: The exact duration of illumination may need to be optimized for specific applications. In this protocol, examples of illumination duration of 30 min, 1 h, 3 h, and 6 h are provided.
4. Remove Petri dish lids to minimize light scattering from condensation.
5. Place Petri dishes containing control embryos in a lightproof box or cover with aluminum foil for dark controls.

3. Quantitative assessment of induction by qRT-PCR

1. Remove embryos from illumination after the desired activation duration.
2. Extract total RNA from 30-50 light-activated and 30-50 dark embryos using an RNA isolation kit following the kit's instructions.
   1. Transfer embryos to a 1.5 mL microcentrifuge tube and remove excess egg water. Add lysis buffer and homogenize the embryos with a plastic pestle.
   2. Transfer the lysate to a kit-provided column and continue with the kit's instructions. Immediately proceed to step 3.3 or store purified RNA at -20 °C to -80 °C.
3. Use 1 µg total RNA for cDNA synthesis using a cDNA synthesis kit following the kit's instructions.
   1. Take a thin-walled 0.2 mL PCR tube, add 1 µg of RNA to 4 µL of 5x cDNA reaction master mix (containing optimized buffer, oligo-dT and random primers, and dNTPs), 1 µL of 20x reverse transcriptase solution, and nuclease-free water to a total volume of 20 µL.
   2. Place the tube in a thermocycler programmed as follows: 22 °C for 10 min, 42 °C for 30 min, 85 °C for 5 min, and then hold at 4 °C. Immediately proceed to step 3.4 or store cDNA at -20 °C.
4. Prepare qPCR reactions containing SYBR green enzyme master mix, 5-fold diluted cDNA (step 3.3), and 325 nM of each primer for GFP.
   NOTE: Primers used in this protocol as follows. GFP forward: 5'-ACGACGGCAACTACAAGCACC-3'; GFP reverse: 5'-GTCCTCCTTGAAGTCGATGC-3'; ef1a forward 5'-CACGGTGACAACATGCTGGAG-3'; ef1a reverse: 5'-CAAGAAGAGTAGTACCGCTAGCAT-3').
5. Carry out qPCR reactions in a real-time PCR machine.
   NOTE: PCR program: initial activation at 95 °C for 10 min, followed by 40 cycles of 30 s at 95 °C, 30 s at 60 °C, and 1 min at 72 °C.
6. Perform a melt curve analysis once the PCR is completed to determine the reaction specificity. Perform three technical replicates for each sample.
7. Calculate light-activated induction as fold change relative to embryos kept in the dark using the 2^-ΔΔCt method¹¹. Statistical significance can be determined with a statistics software package.

4. Qualitative assessment of induction by fluorescence microscopy

1. Remove the embryos from illumination after the desired duration of activation.
2. Immobilize embryos for imaging in 3% methylcellulose containing 0.01% tricaine in glass depression slides.
3. Acquire fluorescence and brightfield images on a fluorescent stereomicroscope connected to a digital camera using standard GFP filter settings. Use identical image acquisition settings for all samples.
4. Merge brightfield and fluorescence images after the acquisition with image processing software.

Results

For this demonstration, a C120-responsive GFP reporter line (Tg(C120F:GFP)^ucm107)) was crossed with a transgenic line that expresses TAEL-N ubiquitously from the ubiquitin b (ubb) promoter (Tg(ubb:TAEL-N)^ucm113)) to produce double transgenic embryos containing both elements. 24 h post-fertilization, the embryos were exposed to activating the blue light, pulsed at a frequency of 1 h on/1 h off. Induction of GFP expression was quantified by qRT-PCR at 30 min, 1 h, 3...

Discussion

This protocol describes the use of the optogenetic TAEL/C120 system to achieve blue light-inducible gene expression. This system consists of a transcriptional activator, TAEL, that dimerizes upon illumination with blue light and activates transcription of a gene of interest downstream of a C120 regulatory element. Induced expression of a GFP reporter can be detected after as little as 30 min of light exposure, suggesting that this approach possesses relatively fast and responsive kinetics.

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Materials

Name	Company	Catalog Number	Comments
BioRender web-based science illustration tool	BioRender		https://biorender.com/
Color CCD digital camera	Lumenara	755-107
Compact Power and Energy Meter Console, Digital 4" LCD	Thorlabs	PM100D
Excitation filter, 545 nm	Olympus	ET545/25x
illustra RNAspin Mini kit	GE Healthcare	95017-491
Instant Ocean Sea Salt	Instant Ocean	SS15-10
MARS AQUA Dimmable 165 W LED Aquarium light (blue and white)	Amazon	B017GWDF7E
Methylcellulose	Sigma-Aldrich	M7140
NEARPOW Programmable digital timer switch	Amazon	B01G6O28NA
PerfeCTa SYBR green fast mix	Quantabio	101414-286
Photoshop image procesing software	Adobe
Prism graphing and statistics software	GraphPad
qScript XLT cDNA SuperMix	Quantabio	10142-786
QuantStudio 3 Real-Time PCR System	Applied Biosystems	A28137
Stereomicroscope	Olympus	SZX16
Tricaine (Ethyl 3-aminobenzoate methanesulfonate)	Sigma-Aldrich	E10521
X-Cite 120 Fluorescence LED light source	Excelitas	010-00326R	Discontinued. It has been replaced with the X-Cite mini+