This protocol is significant because it is an optogenetic, or light-activated method, for inducing gene expression, in an intact organism. In our case, a zebrafish embryo. The advantage of this technique, is that it uses light, as an inducing agent, which allows for very precise, spacial and temporal control of induction.
This protocol would be useful for any situation where inducible gene expression would be useful. This includes things like lineage tracing, gain-of-function assays or rescue experiments. To begin with position the LED panel relative to the Petri dishes containing embryos for delivering 1.5 milliwatts per square centimeter power of light to several embryos at once.
For reducing the risk of photo damage to the Tail Transcriptional Activator, post the light at intervals of one hour on-off using a timer relay, if illuminating for more than three hours. Remove Petri dish lids to minimize light scattering from condensation. Place Petri dishes containing control embryos and a light-proof box for dark controls.
Remove embryos from illumination, after the desired activation duration. For total RNA extraction, use an RNA extraction kit. Transfer the embryos to a 1.5 milliliter micro centrifuge tube and remove the excess egg water.
After adding lysis buffer, homogenize the embryos with the plastic pestle. Transfer the lysate to a kit provided column and follow the manufacturer's instructions to purify RNA. Store the purified RNA, at minus 20 to minus 80 degrees Celsius, until use or immediately synthesize cDNA from one microgram, total RNA using the cDNA synthesis kit, by following the manufacturer's instruction.
Add one microgram of RNA into a thin wall 0.2 milliliter, PCR tube, containing four microliters, of five times cDNA reaction master mix, followed by the addition of one microliter, of 20 times reverse transcriptase enzyme solution. Next make up a final volume of 20 microliters, with nuclease-free water. Place the tube in a programmed Thermo cycler for cDNA synthesis.
Store the synthesized cDNA at minus 20 degrees celsius until use or immediately use for quantitative assessment. Set up the quantitative PCR reactions containing cyber green enzyme master mix. Five fold diluted cDNA and GFP primers at 325 nanomolar concentration in triplicates and conduct the reaction in a programmed real-time PCR machine.
Upon completion of the reaction, analyze the melt curve to determine the reaction specificity. Calculate light-activated induction as fold change relative to the control embryos using the double Delta CT method. And use statistical software to determine the significance.
After removing the embryos from illumination, immobilize embryos for imaging in 3%methylcellulose containing 0.01%Tricaine in glass depression slides. Acquire fluorescence and brightfield images on a fluorescent stereo microscope connected to a digital camera using standard GFP filter settings. Merge brightfield and fluorescence images after the acquisition with image processing software.
Quantification of GFP expression indicated induction of expression as soon as 30 minutes after the blue light exposure. The levels of GFP expression then continued to increase up to six hours, post-activation steadily. GFP induction was also qualitatively assessed by examining the fluorescence intensity at the same time points post-activation.
GFP fluorescence above background levels was first observed at three hours post-activation and became noticeably brighter at six hours Post-activation. In contrast control embryos for all time points, kept in the dark, did not exhibit, any appreciable GFP fluorescence. The activating light must be in the blue range of the visible light spectrum.
Both room light and sunlight contains some blue light. So take care to protect your negative controls from any ambient light sources. The TAEL C120 system, is genetically encoded and modular and can drive expression of any gene of interest.
So, combined with other techniques, it can be used to answer a range of biological questions.
