Assaying NanoLuc Luciferase Activity from mRNA-Injected Xenopus Embryos

Abstract

The earliest steps of animal development depend upon posttranscriptional events that drive the embryonic cell cycle and guide cell fate decisions. The analysis of post-transcriptional regulatory events has relied upon the use of chimeric reporter mRNAs that encode firefly luciferase fused to potential regulatory sequences. A new and more sensitive luciferase developed recently called NanoLuc has the potential to improve reporter studies and provide new insights into the regulation of embryonic processes. Here I describe how to create and analyze reporter mRNAs encoding NanoLuc luciferase using extracts from microinjected Xenopus embryos.

1. Introduction

Developmental biologists have long embraced the use of reporter technologies for their studies. The myriad applications of these tools have provided significant new insights into the fundamental processes that occur in embryonic cells. For example, firefly luciferase-based reporters have been used extensively to quantitatively monitor spatial and temporal gene expression at both the transcriptional and post-transcriptional levels [1–4]. In addition, fluorescent reporters have been used extensively to follow the temporal and spatial expression and activities of specific proteins [5, 6].

The utility and adoption of the different reporters as experimental tools have largely paralleled technical advances. The wide-spread use of firefly luciferase was greatly facilitated by the cloning of the luciferase enzyme and development of sensitive instruments for measuring luciferase activity along with the establishment of assay conditions and substrates that produced prolonged and easily detectible light signals [7, 8]. The identification of fluorescent proteins coupled to the creation of tools to generate specific protein fusions has revolutionized the study of development and significantly advanced our knowledge of key embryonic processes [5, 6]. To further expand the utility of luciferase reporters a new luciferase, called NanoLuc, was developed using directed evolution of an enzyme from the deep-sea shrimp Oplophorus gracilirostris [9]. NanoLuc is a small monomeric protein that utilizes a novel substrate to generate stable luminescence as well as a higher specific activity compared to other luciferase enzymes. This reporter and its unique properties offer numerous advantages for developmental biologists. Particularly, the increased sensitivity of NanoLuc compared to firefly luciferase provides the potential to analyze the activity of reporter mRNAs at concentrations approaching the physiological levels present in embryonic cells [10]. In the following sections I describe how to create and analyze reporter mRNAs encoding NanoLuc luciferase using extracts prepared from microinjected Xenopus embryos.

2. Materials

2.1. Plasmidfor Creating Chimeric NanoLuc mRNAs

To generate mRNAs in vitro for analysis requires a plasmid containing the NanoLuc coding region downstream of a T7 promoter. The plasmids generated by Promega all lack a T7 promoter for generating mRNAs in vitro. To address this issue we created pT7-NanoLuc from pNL1.1 (Fig. 1). In addition to the T7 promoter the XbaI restriction site downstream of the NanoLuc coding region facilitates the cloning of 3ʹUTR fragments for analysis.

Fig. 1.

The pT7-NanoLuc plasmid for creating NanoLuc mRNAs in vitro. The pNL1.1 plasmid (Promega) was modified by cloning the promoter for T7 RNA polymerase upstream of the NanoLuc coding region. An XbaI restriction site is present 30 of NanoLuc and can be used for cloning DNA fragments that encode 30UTRs for analysis

2.2. In Vitro Transcriptionto Generate NanoLuc mRNAs

1. Plasmid for transcription linearized with appropriate restriction enzyme.

2. T7 RNA polymerase.

3. 10×Transcription buffer: 400 mM Tris–HCl, 60 mM MgCl₂, 10 mM DTT, 20 mM spermidine, pH 7.9@25 °C.

4. Nucleotide solutions, ATP 100 mM, CTP 100 mM, UTP 100 mM, GTP 20 mM, CAP analog 40 mM.

5. Nuclease-free water.

6. G50 spin columns.

7. Agarose.

8. 5 ×Tris-borate-EDTA (TBE) buffer (450 mM Tris-borate,10 mM EDTA).

9. RNA-loading dye (47.5% formamide, 0.01% SDS, 0.01% bromophenol blue, 0.005% xylene cyanol, 0.5 mM EDTA).

2.3. Microinjection into Xenopus Embryos

1. Microinjection apparatus.

2. Dissecting microscope.

3. Microinjection dishes with mesh grids to hold embryos in position.

4. Embryo culture media Marc’s Modified Ringer’s Solution (MMR)—10 stock: 1 M NaCl, 20 mM KCl, 10 mM MgSO₄, 20 mM CaCl_2, 50 mM HEPES (pH 7.8), 1 mM EDTA. Adjust pH to 7.4, sterilize by autoclaving, and store at room temperature.

5. MMR 0.25 (working solution).

6. MMR 0.25, 4% Ficoll 400.

2.4. Generating Embryo Extractsfor NanoLuc Assays

1. Luciferase Cell Culture Lysis Reagent 5×(Promega).

2. Micro-pestles.

2.5. Assaying Embryo Extracts for NanoLuc Activity

1. Nano-Glo Luciferase Assay Substrate (Promega).

2. Nano-Glo Luciferase Assay Buffer (Promega).

3. Luminometer assay tubes.

4. Luminometer (for example, for single-tube analysis the Turner Designs 2020 or the Berthold Lumat LB9507 instruments work well). In addition, for the analysis of large number of samples there are a variety of luminometers capable of analyzing samples in microtiter plates.

3. Methods

3.1. Generatethe Plasmid Template for Transcription

1. Plasmid DNA is linearized by restriction enzyme cutting to create a template for in vitro transcription.

2. The linearized DNA template is purified by extraction with an equal volume of 1:1 phenol/chloroform mixture.

3. The DNA is precipitated from the reaction by adding 1/10th volume of 3 M sodium acetate, pH 5.2, and three volumes of ethanol. Incubate at 20 °C for 60 min.

4. The DNA precipitate is spun out of solution using a microcentrifuge operating at 10,000 RPM (10,600 g), for 15 min at 4°.

5. The supernatant is removed and the DNA pellet is rinsed with 500 μL of cold 70% ethanol and centrifuged for 5 min.

6. Remove the wash, air-dry the pellet, and resuspend it in DEPC-treated water at a concentration of approximately 0.5–1.0 μg/μL.

3.2. In Vitro Transcription to Generate NanoLuc mRNAs [11] (See Note 1)

1. Assemble 20 μL reactions at room temperature in the following order (see Note 2):

   Nuclease-free water X μL

   10×Transcription buffer 2 μL

   2 μL ATP 100 mM—10 mM final concentration

   2 μL UTP 100 mM—10 mM final concentration

   2 μL CTP 100 mM—10 mM final concentration

   2 μL GTP 20 mM—2 mM final concentration

   4 μL Cap Analog 40 mM—8 mM final concentration

   Template DNA X μL 1 μg

   T7 RNA polymerase 2 μL

   Total reaction volume 20 μL

2. Mix by micropipetting and incubate at 37 °C for 2 h.

3. Add nuclease-free H₂O to the reaction until the volume reaches 100 μL.

4. G50 spin columns are used to remove unincorporated nucleotides, proteins, and salts. Apply the reactions to the column and follow the manufacturer’s instructions for sample purification.

5. Measure ultraviolet light absorbance at 260 nm (see Note 3).

6. Analyze an aliquot of the mRNA on a denaturing agarose gel to evaluate length, integrity, and quality [12, 13] (see Note 4).

3.3. Microinjection into Xenopus Embryos

1. Generate Xenopus embryos and microinject reporter mRNAs using standard protocols [14–17] (see Note 5).

2. Incubate injected embryos in 0.25MMR 4% Ficoll until the desired stage of development is reached (see Note 6).

3.4. Generating Embryo Extractsfor NanoLuc Assays

1. Examine injected embryos and discard any damage during microinjection.

2. Place healthy embryos in 1.5 mL microcentrifuge tubes.

3. Remove excess culture media taking care not to lyse the embryos.

4. Add 100 μL cell culture lysis reagent to each embryo sample (see Note 7).

5. Lyse the injected cells with a micro-pestle.

6. Spin the extracts at 4 °C for 10 min at 10,000 RPM (10,600 g) in a microfuge.

7. Transfer the supernatant extracts to a new microcentrifuge tube while leaving behind the particulate pellets and yolk.

8. Assay extracts for NanoLuc activity immediately or store extracts at 80 °C indefinitely (see Notes 8 and 9).

3.5. Assaying Embryo Extracts for NanoLuc Activity

1. Thaw extracts on ice or if generated fresh store on ice until use (see Notes 8 and 9).

2. Prepare a working solution of the NanoLuc substrate by diluting the concentrated substrate 1/50 in NanoLuc assay buffer (see Note 10).

3. Prepare samples by mixing 50 μL of substrate with 50 μL of extract and incubating at room temperature for 3 min protected from light (see Note 11).

4. Transfer samples to luciferase assay tubes and assay for NanoLuc activity using a luminometer (see Notes 12 and 13).

4. Notes

1. Follow precautions for working with mRNA to avoid RNase contamination that could degrade the mRNA. Wear gloves at all times and use only nuclease-free tubes and reagents.

2. Reactions are typically 20 μL but can be scaled up if large amounts of mRNA are needed.

3. Calculate mRNA concentration, with one A260 unit being equivalent to an mRNA concentration of 40 μL/mL.

4. Mix 0.2 μg mRNA sample with RNA-loading dye. Denature the mRNA sample by heating at 65 °C for 5 min. Load sample onto a 1% agarose gel (TBE buffered) and electrophorese. Visualize RNA by staining the gel with ethidium bromide.

5. Concentrations of mRNAs in samples for injection will vary depending upon individual mRNA and specific application. Typically we use samples that contain 6 attomoles/nL (1 pg/nL) mRNA and inject 1–10 nL per embryo [14–16].

6. Embryos can be cultured at lower temperatures approaching 13 °C to slow development when collecting samples for time-course experiments.

7. The volume of cell culture lysis buffer is typically 100 μL/embryo but the volume can be varied to accommodate different signal intensities.

8. Extracts can be assayed immediately after production or stored at 80 °C and assayed when convenient.

9. We have not observed a reduction of activity upon freezing and thawing extracts multiple cycles.

10. Each assay requires 50 μL of substrate. The prepared substrate is kept at room temperature protected from light until use.

11. Assay each extract in triplicate to provide technical replicates. Analyze multiple dilutions of each extract to ensure that readings are in the linear range of the assay conditions and the luminometer. Prepare dilutions of extract with cell culture lysis buffer. Once prepared the cell culture lysis buffer can be stored at 20°until use.

12. Occasionally negative control extracts from uninjected embryos produce a background signal (>1000 RLUs). In such cases freezing and thawing extract often eliminate the background.

13. Although this chapter focuses on analyzing NanoLuc activities in Xenopus embryos similar approaches can be applied to Xenopus oocytes, eggs, and embryos of other model organisms used by developmental biologists.