This protocol describes an optogenetic strategy to modulate mitogen-activated protein kinase (MAPK) activity during cell differentiation and Xenopus embryonic development. This method allows for the reversible activation of the MAPK signaling pathway in mammalian cell culture and in multicellular live organisms, like Xenopus embryos, with high spatial and temporal resolution.
Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early embryonic development, kinase activity is highly dynamic and widespread across the embryo. Pharmacological and genetic approaches are commonly used to probe kinase activities. Unfortunately, it is challenging to achieve superior spatial and temporal resolution using these strategies. Furthermore, it is not feasible to control the kinase activity in a reversible fashion in live cells and multicellular organisms. Such a limitation remains a bottleneck for achieving a quantitative understanding of kinase activity during development and differentiation. This work presents an optogenetic strategy that takes advantage of a bicistronic system containing photoactivatable proteins Arabidopsis thaliana cryptochrome 2 (CRY2) and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). Reversible activation of the mitogen-activated protein kinase (MAPK) signaling pathway is achieved through light-mediated protein translocation in live cells. This approach can be applied to mammalian cell cultures and live vertebrate embryos. This bicistronic system can be generalized to control the activity of other kinases with similar activation mechanisms and can be applied to other model systems.
Growth factors are involved in a wide spectrum of cell functions, including proliferation, differentiation, migration, and apoptosis, and play pivotal roles in many biological events, including embryonic development, aging, and regulation of mental status1,2,3,4,5. Many growth factors signal through complex intracellular signaling cascades. These signaling events are often operated by reversible protein phosphorylation in a precisely regulated fashion6,7. Thus, an understanding of the signaling outcomes of protein kinases, which are responsible for protein phosphorylation, is fundamentally important.
Different growth factors act through a rather common intracellular signaling network, even though they stimulate distinct cellular responses8,9. Common intracellular mediators of receptor tyrosine kinases include Ras, Raf, extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase (MAPK)/ERK kinase (MEK), phosphoinositide 3-kinase (PI3K), Akt, and phospholipase C gamma (PLCγ)10,11. Accumulating evidence suggests that signaling diversity and specificity depend upon the spatial and temporal regulation of signaling activity12. For instance, in rat pheochromocytoma cells (PC12), epidermal growth factor (EGF) stimulation, which results in cell proliferation, transiently activates the ERK pathway9. On the other hand, stimulation with nerve growth factor (NGF), which leads to cell differentiation, activates the ERK pathway in a sustained manner9,13. In cultured rat hippocampal neurons, transient signaling by brain-derived neurotrophic factor (BDNF) promotes primary neurite outgrowth, while sustained signaling leads to increased neurite branching14. During early embryonic development, phosphorylated ERK activity is temporally dynamic and is widespread across the embryo6. A recent genetic screen during early Xenopus embryogenesis showed that ERK and Akt signaling cascades, two downstream primary growth factor pathways, display stage-specific activation profiles7. Thus, an understanding of kinase signaling outcomes calls for tools that can probe the spatial and temporal features of kinase activity with sufficient resolution.
Conventional experimental approaches to probe the dynamic nature of signal transduction during development lack the desirable spatial and temporal resolution. For instance, pharmacological approaches utilize small chemical or biological molecules to stimulate or suppress signal transduction in cells and tissues. The diffusive nature of these small molecules makes it challenging to restrict their action to a specific region of interest15. Genetic approaches (e.g., transgenesis, the Cre-Lox system, or mutagenesis) often lead to the irreversible activation or repression of the target gene expression or protein activity16,17,18. The Tet-On/Tet-Off system19 offers improved temporal control of gene transcription but lacks strict spatial control because it relies on the diffusion of tetracycline. Recent developments in chemically induced protein dimerization20 or photo-uncaging21,22,23,24 have greatly enhanced the temporal control of signaling networks. The spatial control, however, remains challenging due to the diffusive nature of the caged chemicals.
Recent emerging optogenetic approaches, which harness the power of light to control protein-protein interactions, allow for the modulation of signaling pathways with high spatiotemporal precision as well as reversibility. Shortly after its initial success in controlling neuronal firing25,26,27, optogenetics has been extended to control other cellular processes, such as gene transcription, translation, cell migration, differentiation, and apoptosis28,29,30,31,32,33,34. A strategy using the photoactivatable protein pair Arabidopsis thaliana cryptochrome 2 (CRY2) protein and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN) was recently developed to control Raf1 kinase activity in mammalian cells and Xenopus embryos35. CRY2 binds to CIBN upon blue-light stimulation, and the CRY2/CIBN protein complex dissociates spontaneously in the dark34. Blue light excites the CRY2 cofactor, flavin adenine dinucleotide (FAD), which leads to a conformational change in CRY2 and its subsequent binding to CIBN. Constitutively active (W374A) and flavin-deficient (D387A) mutants of CRY2 can be produced through mutations in the FAD-binding pocket: the CRY2W374A mutant binds to CIBN independent of light, whereas the CRY2D387A mutant does not bind to CIBN under blue-light stimulation36,37. The optogenetic system described in this protocol uses wild-type CRY2 and CIBN to induce protein translocation-mediated Raf1 activation in live cells. It is known that the membrane recruitment of Raf1 enhances its activity38. In this system, a tandem CIBN module is anchored to the plasma membrane and CRY2-mCherry is fused to the N-terminal of Raf135. In the absence of blue light, CRY2-mCherry-Raf1 stays in the cytoplasm, and Raf1 is inactive. Blue-light stimulation induces CRY2-CIBN binding and recruits Raf1 to the plasma membrane, where Raf1 is activated. Raf activation stimulates a Raf/MEK/ERK signaling cascade. Both CRY2- and CIBN- fusion proteins are encoded in a bicistronic genetic system. This strategy can be generalized to control other kinases, such as Akt, whose activation state can also be turned on by protein translocation in cells39. This work presents detailed protocols for implementing this optogenetic strategy in mammalian cell cultures and multicellular organisms.
Animal research was conducted in accordance with guidelines set by the Illinois Institutional Animal Care and Use Committee (IACUC) and the University of Illinois Department of Animal Resources (DAR).
1. Optogenetic Induction of Protein Localization in BHK21 Mammalian Cell Culture
NOTE: Steps 1.1-1.3 provide a method to assemble a cell culture chamber for imaging with high-magnification objectives (e.g., 63X or 100X), which typically have short working distances. These objectives require a thin glass coverslip (e.g., #1.5, 170 µm thickness) as the imaging substrate. Alternatively, a glass-bottom cell culture dish/slide can be used. In such a case, steps 1.1-1.3 can be skipped.
2. Construction of an LED Array for Long-term Light Stimulation in a CO2 Incubator
NOTE: The overall schematic of the experimental setup is shown in Figure 2A.
3. Optogenetic Induction of PC12 Cell Differentiation
4. Optogenetic Control of Kinase Activity in Xenopus Embryos
Ratiometric expression of photoactivatable protein pairs: Figure 1A shows the design of a bicistronic optogenetic construct, CRY2-mCherry-Raf1-P2A-CIBN-CIBN-GFP-CaaX (referred to as CRY2-2A-2CIBN), based on the porcine teschovirum-1 2A (P2A) peptide, which shows the highest ribosome-skipping efficiency among mammalian cell lines42. In previous work, it has been determined that the optimal ratio for CIBN-GFP-CaaX:CRY2-mCherry-Raf1 is 2:135. This configuration allows for sufficient membrane recruitment and activation of CRY2-mCherry-Raf1 (Figure 1B). Cells singly transfected with CRY2-2A-2CIBN show clear fluorescence in both the GFP and Txred fluorescent channels, either under epi-illumination (Figure 1C) or confocal microscopy (Figure 1D). Blue light-mediated membrane recruitment of CRY2-mCherry-Raf1 (Figure 1D) can be clearly detected in confocal microscopy. The association occurs within seconds after blue-light exposure (Figure 1E), and the CRY2-CIBN protein complex dissociates with a half-life of about 5.5 min (Figure 1F).
Light-controlled PC12 cell differentiation in the absence of nerve growth factor: An intriguing observation in growth factor signaling is that common downstream signaling pathways (e.g., ERK, PI3K-Akt, and PLCγ) elicit diversity and specificity in signaling responses. A better understanding of growth factor-mediated signaling can be achieved by enabling technology that allows for the precise spatiotemporal regulation of individual cascades. The optogenetic approach introduced in this protocol demonstrates one such technology that allows for the specific activation of the Raf/MEK/ERK signaling pathway with high temporal resolution. Because this approach bypasses the process of NGF binding to its membrane receptors, the signaling kinetics no longer depend on the endogenous receptors. Instead, precise kinetic control can be achieved by modulating the temporal profile of the stimulating light (Figure 2A). Thus, this approach opens up new possibilities to study the signaling kinetics of kinase activity. Additionally, this experimental setup provides an extremely simple and economic way to integrate the optogenetic approach into studies of intracellular signal transduction (Figure 2B-2D). For the PC12 cell differentiation assay, a 24-h continuous-light stimulation at 0.2 mW/cm2 is sufficient to induce significant neurite outgrowth (Figure 3A). Negative controls, including transfected cells without light (Figure 3B) and non-transfected cells with or without light (Figure 3C-3D), do not show significant neurite outgrowth. At this power, cells transfected with a constitutively active Raf1 (CA-Raf1) undergo normal differentiation (Figure 3E). Notably, cells transfected with the bicistronic optogenetic construct produce a significantly higher differentiation ratio than cells co-transfected with two constructs, reaching the value induced by CA-Raf1 (Figure 3F). The differentiation ratio is calculated by dividing the number of differentiated cells by the number of transfected cells guided by GFP fluorescence. Differentiated cells are defined as those with at least one neurite longer than the size of the cell body35. This enhancement in the differentiation ratio arises from: 1) a better delivery of photoactivatable proteins with the bicistronic system and 2) an optimized expression ratio between CIBN and CRY235.
The reversible optogenetic stimulation of the Raf/MEK/ERK signaling pathway in live Xenopus laevis embryos: X. laevis is a well-established model organism for studying gene transcription, signal transduction, and embryonic development. Previous work discovered that the activation of the Raf/MEK/ERK pathway leads to a cell fate change, resulting in the formation of an ectopic tail-like structure at the tailbud stage43. As a potent mesoderm inducer, overexpressed Raf1 can induce ectopic mesoderm during germ-layer specification, which occurs during the blastula and early gastrula stages, and result in the formation of ectopic tail-like structures later on. Alternatively, overexpressed Raf1 may trigger an epithelial-mesenchymal transition (EMT)-like event after germ-layer specification has completed and can directly transform ectoderm to neural and mesoderm lineages. This is followed by the proliferation and extension of these structures43. In these experiments, RNAs encoding MET receptor, Ras, and Raf1 were injected into embryos at the two-cell stage. Shortly after the RNA was injected into the embryo, the overexpressed MET/Ras/Raf1 began to constitutively activate the downstream signaling cascades. Therefore, it was technically challenging to bypass early developmental stages and activate Raf1 specifically after germ-layer specification. It was impossible to use such a strategy to determine if activation of the Raf/MEK/ERK signaling after germ-layer specification would induce a cell fate change, leading to the formation of the ectopic tail-like structure.
Optogenetics provides a mechanism to control the timing of Raf1 activity. When the RNA of CRY2-2A-2CIBN was injected into embryos at early developmental stages, Raf1 remained inactive until blue light was supplied. The animal cap assay was conveniently used to characterize the signaling outcome, because the basal ERK activity is low in animal caps. The light-mediated activation of the Raf/MEK/ERK signaling cascade induced a significant upregulation of xbra, a mesodermal marker, as determined by RT-PCR (Figure 4A) or whole-mount in situ hybridization (Figure 4B). Dynamic changes in the phosphorylation of ERK in response to blue light were also detected by Western-blot analysis (Figure 4C). In whole embryos, a mixture of CRY2-2A-2CIBN and n-β-gal RNAs was injected at the 8-cell stage into one of the dorsal animal blastomeres, which later give rise to dorsal anterior tissue. Compared with untreated embryos (Figure 4D-4E), those injected with CRY2-2A-2CIBN and treated with blue light formed ectopic tail-like structures (Figure 4F-4G). Injected embryos in the dark (Figure 4H) or uninjected under blue-light illumination (Figure 4I) did not form tail-like structure. Blue-light illumination after germ-layer specification induced significant tail-like structures (Figure 4J).
Figure 1: The mechanism of light-induced protein localization and activation of the kinase signaling pathway. (A) Design of a bicistronic construct, with an optimized CIBN-to-CRY2 ratio (2 CIBN:1 CRY2) that allows for the light-induced activation of the Raf/MEK/ERK kinase signaling pathway. Upon ribosomal skipping, one mRNA transcript generates two proteins: CRY2-mCherry-Raf1-N2A (ending with amino acids NPG) and proline-2CIBN-GFP-CaaX. (B) Light-induced binding between CIBN and CRY2 leads to membrane recruitment of CRY2-mCherry-Raf1, which activates the Raf/MEK/ERK signaling pathway. (C) Fluorescence images of BHK21 cells transfected with CRY2-2A-2CIBN under epi-illumination. Scale bar: 20 µm. (D) Confocal fluorescence imaging of cells transfected with CRY2-2A-2CIBN. The left panel shows cleaved 2CIBN-GFP-CaaX localized on the plasma membrane; the middle panel shows a snapshot of CRY2-mCherry-Raf1 before blue-light stimulation; the right panel shows a snapshot of CRY2-mCherry-Raf1 after 10 pulses of blue-light stimulation. The bottom panel shows normalized intensity profiles along a yellow dotted line across the cell, before and after light stimulation. Scale bar = 20 µm. (E–F) Kinetics for light-induced CRY2-CIBN association (E) and spontaneous dissociation (F) of the CRY2-CIBN protein complex in the dark. Figure 1A-B was adapted from reference35, reproduced with the permission of the Company of Biologists. Figure 1E-F was adapted from reference44 under the Creative Commons Attribution (CC BY) license. Please click here to view a larger version of this figure.
Figure 2: Design and calibration of the home-built LED array. (A) Schematic of the experimental setup for the light-controlled kinase activity in live cells. (B) Circuit board for the home-built LED array. (C) A light box that can be used for long-term light stimulation in a CO2 incubator. The height of the aluminum box is 2 in. (D) Typical LED output power versus voltage. Values light power per LED in a circuit in which a current-limiting resistor and four LEDs are connected in series. Figure 2A was adapted from reference35, reproduced with the permission of the Company of Biologists. Please click here to view a larger version of this figure.
Figure 3: Optogenetic induction of PC12 cell differentiation. (A) Multi-channel snapshots of PC12 cells transfected with CRY2-2A-2CIBN after 24 h of blue-light stimulation (0.2 mW/cm2). A circle marks a differentiated cell. A square marks an undifferentiated cell. (B) Same as (A), except no blue-light stimulation was used. (C–D) Non-transfected PC12 cells under 24 h of blue-light stimulation (0.2 mW/cm2) (C) or dark incubation (D). (E) Representative images of cells transfected with Raf1-GFP-CaaX (CA-Raf1). The circle and rectangle mark differentiated and undifferentiated cells, respectively. (F) Differentiation ratios of PC12 cells transfected with CA-Raf1, co-transfected with CRY2-mCherry-Raf1 and CIBN-GFP-CaaX, and singly transfected with CRY2-2A-2CIBN. 24 h after transfection, cells were either exposed to light or incubated in the dark for another 24 h. The values represent the mean ± SD from four independent data sets. Only cells exposed to blue light showed significant differentiation. Figure 3F was adapted from reference35, reproduced with the permission of the Company of Biologists. Please click here to view a larger version of this figure.
Figure 4: Optogenetic induction of kinase activity in live Xenopus embryos. (A) RT-PCR results showing that exposure to blue light induced the expression of xbra, a pan-mesodermal marker, in CRY2-2A-2CIBN-injected animal caps at the early gastrula stage. (B) Whole-mount in situ hybridization of xbra in embryos. Light-induced activation of MAPK activity modulates the spatial distribution of xbra (red arrows in the bottom-right panel). Black arrows mark nuclear β-galactosidase as the lineage tracer. AP: animal pole. VP: vegetal pole. (C) Western-blot analysis showing that, in an animal cap assay, CRY2-2A-2CIBN induced blue light-dependent, reversible phosphorylation of ERK. (D) Images showing the morphology of normal embryos, without mRNA injection and without light treatments. (E) A zoomed-in image of a single embryo. (F–G) Zoomed-in and whole-field images for embryos injected with CRY2-2A-2CIBN mRNA and subjected to blue-light stimulation. Activation of Raf1 by treating CRY2-2A-2CIBN-injected embryos with blue light induces ectopic tail-like structures in the head region. (H–I) Negative controls, including embryos injected with CRY2-2A-2CIBN but under dark incubation (H) and uninjected embryos under light stimulation (I). All light stimulation experiments were conducted with a power of 5 mW/cm2. (J) Statistical analysis of the percentage of embryos showing ectopic tail-like structure under each condition. Scale bar = (D) and (G): 1 mm; (E–F) and (H–I) 0.5 mm. Figure 4A, C, E–F, and H–J were adapted from reference35, reproduced with the permission of the Company of Biologists. Please click here to view a larger version of this figure.
When building the light box, the power of individual LEDs should be measured. Based on previous experience, the power output can vary between individual LEDs due to manufacturing variance. Select a set of LEDs that have a power output within 10% of each other. The number of LEDs, the current-limiting resistor, and the power input can be modified for different types of cell culture containers (e.g., a 6-well or 24-well plate). A 24 h of light illumination at a power of 0.2 mW/cm2 does not induce detectable phototoxicity44. If a higher power is used, consider using intermittent light to reduce heat generation and phototoxicity. The optogenetic system introduced in this protocol induces neurite outgrowth in PC12 cells under intermittent light stimulation comparable to that of continuous light stimulation, given that the dark interval between the two adjacent light pulses is less than 45 min44. Due to intrinsic differences in the kinetics of protein association and dissociation, the duration between light pulses must be tuned for each unique protein pair. Overexpression of cytosolic Raf1 does not cause PC12 cell differentiation without blue-light illumination. By controlling the amount of mRNA injected into each embryo, no significant embryonic phenotypes were observed in the dark.
Although a low-power blue light is sufficient to stimulate the association of CRY2- and CIBN-fusion proteins, the poor penetration depth of blue light limits its use in deep tissue stimulation. Although such stimulation has been achieved by delivering light via other devices (e.g., fiber optics), the approaches are invasive45. This issue can be addressed in two ways: by using a protein pair that responds to longer wavelength (e.g., the phytochrome-PIF6 protein pair) or by using two-photon excitation. Both approaches can provide deeper penetration, but they could suffer from other limitations. For example, the PhyB-PIF6 pair requires a synthetic cofactor to function, and two-photon microscopy requires costly instrumentation. The lack of tunability in the CRY2-CIBN interaction is another limitation to consider. Wild-type CRY2 spontaneously dissociates from CIBN with a half-life of 5.5 min in the dark34. Depending on the kinetics of the target signaling pathway, a shortened or prolonged dissociation half-life may be preferred. Mutations in CRY2 have been made that can either shorten the dissociation half-life to 2.5 min (W349R) or prolong it to 24 min (L348F)46. Alternatively, engineered photoactivatable protein pairs based on fungal receptor Vivid (VVD) and p/nMagFast1 and 2 show a dissociation half-life of 4.2 min and 25 s, respectively47. In this protocol, the wild-type CRY2-CIBN protein pair turns on the MAPK pathway within 5 min of light stimulation, and the activity decays back to the basal level within 30 min in mammalian cells44 and 1-2 h in Xenopus embryos35. For spatial control, confocal microscopy can focus the blue laser to the size of a diffraction-limit spot of about 200 nm in the image plane. By adjusting the size of field diagram in an epi-illumination microscope equipped with a non-coherent light source, it is possible to limit the illumination size to about 10 µm in diameter. Both methods should be able to achieve the subcellular control of optogenetic stimulation in cell cultures.
The ability of optogenetics to reversibly interrogate signal transduction with high spatial and temporal resolution offers an entirely new opportunity to study dynamic intracellular signaling in live cells. Results from this protocol have revealed that Raf1 activation is primarily responsible for the induction of neuronal differentiation in PC12 cells44. The same pathway also causes the formation of secondary tail-like structures in developing Xenopus embryos. By controlling the timing of Raf1 activation, the tail-like structure can be induced after the stage of germ-layer formation35. The recently described LOVTRAP method utilizes two engineered proteins, Zdark and LOV2, which selectively bind to each other only in the dark to modulate the light-induced dissociation of a protein of interest (POI)48. Unlike light-mediated protein-protein association, which is used in this approach, LOVTRAP uses light to induce protein dissociation. This new modality is more flexible at modulating protein localization and activity in live cells. By carefully selecting the mode of activation within a signaling cascade, it is possible to dissect signal transduction in unconventional ways. For instance, it is possible to activate receptor tyrosine kinases without ligand-receptor binding49 or to induce a subset of ligand-elicited signaling cascades44. The strategy reported here can be generalized to control other kinases, such as Akt39 and GTPases (e.g., Rho GTPase), whose activation states can also be turned on by protein translocation. Optogenetics will continue to be applied to live multicellular organisms, as demonstrated by recent works featuring the optogenetic control of protein localization and signaling in Drosophila50, zebrafish51,52,53,54, and Xenopus embryos35.
The authors have nothing to disclose.
This work was supported by the University of Illinois at Urbana-Champaign (UIUC) and the National Institutes of Health (NIGMS R01GM111816).
Glass coverslip | VWR | 48393 230 | Substrate for live cell imaging |
Coverslip holder | Newcomer Supply | 6817B | Holder for coverslips |
Detergent | ThermoFisher | 16 000 104 | For cleaning coverslips |
Boric acid | Sigma-Aldrich | B6768-500G | For making PLL buffer |
Disodium tetraborate | Sigma-Aldrich | 71996-250G | For making PLL buffer |
Plastic beaker | Nalgene | 1201-1000 | For cleaning coverslips |
Sodium hydroxide | Sigma-Aldrich | 221465-2.5KG | For adjust pH |
Poly-L-lysine hydrobromide | Sigma-Aldrich | P1274-500MG | For coating coverslip |
Diethylpyrocarbonate (DEPC)-Treated Water | ThermoFisher Scientific | 750024 | For DNA preparation |
Cover Glass Forceps | Ted Pella | 5645 | Cover glass handling |
Tissue cutlure dish | Thermofisher | 12565321 | Cell culture dish |
Sterile centrifuge tubes | ThermoFisher | 12-565-271 | Buffer storage |
Transfection Reagent | ThermoFisher | R0534 | Transfection |
CO2-independent medium | ThermoFisher | 18045088 | For live cell imaging |
Polydimethylsiloxane (PDMS) | Ellsworth Adhesives | 184 SIL ELAST KIT 0.5KG | Form make cell chamber |
Plasmid Maxiprep kit | Qiagen | 12965 | Plasmid preparation |
DMEM medium | ThermoFisher | 11965-084 | Cell culturing medium component |
F12K medium | ThermoFisher | 21127022 | Cell culturing medium component |
Horse serum | ThermoFisher | 16050122 | Cell culturing medium component |
Fetal Bovine Serum | Signa-Aldrich | 12303C-500 mL | Cell culturing medium component |
Penicillin-Streptomycin-Glutamine | ThermoFisher | 10378016 | Cell culturing medium component |
Trypsin (0.25%), phenol red | ThermoFisher Scientific | 15050065 | For mammalian cell dissociation |
Agarose | Fisher Scientific | BP1356-100 | For DNA preparation |
Ficoll PM400 | GE Heathcare Life Sciences | 17-5442-02 | For embryo buffer |
L-Cysteine hydrochloride monohydrate | Sigma-Aldrich | 1.02839.0025 | Oocyte preparation |
ApaI | ThermoFisher | FD1414 | For linearization of plasmids |
Dnase I | ThermoFisher | AM2222 | For removing DNA template in the in vitro transcription assay |
Index-match materials (immersion oil) | Thorlabs | MOIL-20LN | For matching the index between sample substrate and objective |
Blue LED | Adafruit | 301 | Light source for optogenetic stimulation |
Resistor kit | Amazon | EPC-103 | current-limiting resistor |
Aluminum boxes | BUD Industries | AC-401 | light box |
BreadBoard | Jekewin | 837654333686 | For making LED array |
Hook up Wire | Electronix Express | 27WK22SLD25 | For making LED array |
Relay Module | Jbtek | SRD-05VDC-SL-C | For intermittent light control |
DC Power Supply | TMS | DCPowerSupply-LW-(PS-305D) | Power supply for LED |
Silicon Power Head | Thorlabs | S121C | For light intensity measurement |
Power meter | Thorlabs | PM100D | For light intensity measurement |
Microscope | Leica Biosystems | DMI8 | For live cell imaging |
BioSafety Cabinet | ThermoFisher | 1300 Series A2 | For mammalian cell handling |
CO2 incubator | ThermoFisher | Isotemp | For mammalian cell culturing |
Stereo microscope | Leica | M60 | For embryo micro-manipulation |
Microinjector | Narishige | IM300 | For embryo microinjection |
Micropipette puller | Sutter Instruments | P87 | Needle puller |
in vitro transcription kit | ThermoFisher | AM1340 | For in vitro transcription. The kit includes nuclease-free water, SP6 RNA Polymerase, ribonucleotide mixture, cap analog, lithium choride precipitation solution, and spin column |
RNA purfication kit | Qiagen | 74104 | Silica-membrane spin column for purification of synthesized RNA |
Convection oven | MTI corporation | EQ-DHG-9015 | PDMS curing |
Centrifugal mixer and teflon container | THINKY | AR310 | For mixing PDMS |
Silicon wafer | UniversityWafer | 452 | Base for making PDMS devices |
Blade | Techni Edge | 01-801 | For cutting PDMS |
Capillary glass | Sutter Instruments | BF100-58-10 | For fabrication of injecting needles. |