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Method Article

Cell-Free In Vitro mRNA Translation In Drosophila Lysates

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DOI:

10.3791/70844

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June 12th, 2026

 ,  ,  ,  , 
1mRNA and Cancer Laboratory, Subdirección de Investigación Básica, Instituto Nacional de Cancerología (INCan; National Institute of Cancer), 2Escuela de Medicina y Ciencias de la Salud, Tecnologico de Monterrey, 3Escuela de Ingeniería y Ciencias, Tecnologico de Monterrey

In This Article

Summary

Here, we provide details on how to prepare lysates and perform in vitro translation using Drosophila embryos.

Abstract

Cell-free mRNA in vitro translation has played a crucial role in the understanding of the protein synthesis process during gene expression across eukaryotes. The development of lysates from different systems for in vitro translation has been instrumental in studying the roles of most components of the translation machinery and in dissecting many steps of the protein synthesis process. Different aspects of translation and translational control have been studied using Drosophila melanogaster lysates. This paper provides detailed protocols for preparing synthetic mRNA templates, growing large-scale living fly cultures, preparing translation-competent lysates from Drosophila embryos, and performing cell-free in vitro translation reactions. This protocol is suitable for any species of the Drosophila genus—melanogaster, virilis, pseudoscura, grimshawi, hydei, etc. We further compare this protocol to other translation systems and discuss limitations, critical steps, and troubleshooting. Representative outcomes are included to help assess protocol performance and reproducibility. Finally, we discuss potential applications in biotechnology and research.

Introduction

For over a century, Drosophila has proven to be a powerful, genetically tractable model organism to study many biological phenomena and molecules, including translational control and the translation machinery1,2,3,4. Drosophila cell-free in vitro translation has become a potent tool to study protein synthesis, because it can recapitulate many events observed in vivo. Thus, Drosophila is an ideal system for discovering novel functions and molecules performing translation and translational control. Early studies established an in vitro, translationally competent system in the fly-related arthropod Artemia salina5 and later in Drosophila hydei6.

Later, lysates responsive to exogenous mRNAs were developed in Drosophila melanogaster. Most studies on Drosophila in vitro translation have been performed in embryos from different ages, although some variations of the translation reaction buffer have been used by different research groups. For comparison, the different phenomena studied and the translation reaction conditions are listed in Table 1. Besides embryos, protocols for cell-free in vitro translation using Drosophila oocytes10,22,23,24,25,26 and cultured Schneider or Kc cells lysates have also been developed17,27,28,29,30,31,32. A description of the components required for in vitro translation and a comparison among lysates from different species are described in references33,34.

Although the process of mRNA translation is conserved across eukaryotes, significant differences among taxa are found mostly at the initiation step of translation where various molecules repress or promote translation throughout development in a taxon- or species-specific manner35,36. This protocol is very suitable to study mRNA translation in insects throughout development, for which lysates from phylogenetically distant Metazoan cannot be used, including the commercial lysates from wheat or rabbit. Indeed, this protocol can recapitulate the translation events that occur during embryogenesis, something not possible when using oocyte lysates23. To date, no comparison between embryo, oocyte and cultured-cell Drosophila systems has been done. Adding posttranslational modifications to the in vitro synthesized proteins is a limitation of this protocol in its current state. Moreover, this method does not yield preparative amounts of protein compared to methods for producing recombinant proteins in bacteria.

Here, we describe detailed protocols to routinely grow large-scale cages of Drosophila flies, to prepare synthetic mRNA templates, to prepare translation-competent lysates from embryos, and to perform cell-free in vitro translation assays. The Table of Materials shows the reagents required throughout the protocol.

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Protocol

1) Template plasmid construction for in vitro transcription

  1. Select plasmids containing a 5´ untranslated region (UTR), a reporter cistron, a 3´ UTR, and a poly(A) tail downstream of a T7, T3, or SP6 RNA polymerase promoter. Ensure that each construct contains a unique restriction site immediately downstream of the poly(A) tail for plasmid linearization before transcription (Figure 1).
    NOTE: The restriction enzyme site used should be carefully chosen so that the recognition site is outside of the transcribed sequence or one that allows the linearized DNA to end in A. Moreover, the restriction enzyme should leave a blunt or 5' overhang at the 3' end of the template. 3' overhangs can result in aberrant transcription products. If an enzyme with a 3' overhang is used, the fragment should be blunted before use.
  2. Verify all constructs by sequencing before using them as templates for in vitro transcription.
    NOTE: Firefly luciferase (FLuc) reporter constructs with a poly(A)71 tail have been used for this workflow. The FLuc constructs are derived from the pLUC-cassette12. Vector and kit information are listed in the Table of Materials.

2) In vitro transcription of capped and polyadenylated mRNAs

  1. Linearize each plasmid construct using the unique restriction site downstream of the poly(A) tail (here, KpnI).
  2. Resolve the linearized DNA by 1%-agarose gel electrophoresis run at 100 V for 1 h.
  3. Gel-extract the DNA and check its integrity and proper linearization by identical agarose gel electrophoresis.
    NOTE: It is not necessary to gel-purify the linearized DNA—if the DNA is completely cut, then a column purification or phenol-extraction/ETOH precipitation is sufficient.
  4. Transcribe the purified linearized DNA using an in vitro transcription kit according to the manufacturer’s instructions. Include a 5´ cap analog in the transcription reaction to stabilize the RNA and stimulate translation.
    NOTE: A functional cap analog, such as m7G(5′)ppp(5′)G RNA, supports transcript stabilization and translation stimulation. A non-functional cap analog, such as G(5′)ppp(5′)A, supports transcript stabilization but not translation stimulation. This inhibitor of translation is used when bicistronic transcripts are translated to test for the presence of internal ribosome entry sites (IRES) driving translation of the second cistron7. To test for the dependence of a poly(A) tail, this can be deleted in the template plasmid before transcription is performed.
  5. Treat all the transcription reactions with RNase-free DNase to remove the DNA template after transcription according to the manufacturer’s instructions. All in vitro transcription kits provide the RNase-free DNase.
  6. Purify the transcript by lithium chloride precipitation or column-based RNA cleanup according to the manufacturer’s instructions.
    NOTE: All in vitro transcription kits provide the lithium chloride reagents.
  7. Quantify by spectroscopy the purified transcript and assess mRNA integrity by 1%-agarose gel electrophoresis run at 100 V for 1 h.
  8. Aliquot the mRNA and store at -70 °C until use.

3) Routine maintenance of a large-scale Drosophila culture

  1. Use the reference Drosophila melanogaster wild-type strain Oregon-R.
  2. Prepare fly food according to an established reference-center recipe.
    NOTE: The authors used the Bloomington Drosophila Stock Center recipe37 as the food preparation reference. Details of this recipe are given in Table 237. Figure 2 shows the overall workflow from embryo collection through luciferase activity quantitation.
  3. Set a cubic or cylindrical acrylic cage approximately 1 m long and 50 cm in diameter.
  4. Make an approximately 30 cm diameter hole in the upper part of the cage and seal it with mesh (Figure 3).
  5. Place wet paper on top of the mesh to provide water for the flies, and replace the wet paper every day (Figure 3).
  6. Keep the front part of the cage open and covered with fabric to allow access to the inside of the cage. Place acrylic on the back side of the plastic fly cage, totally sealed.
  7. Amplify the fly stock before embryo collection, and place ~1,000 young flies inside the cage. Maintain the flies' cage at 22–25 °C constantly.
    NOTE: Several fly cages of the same dimensions and fly density can be maintained at the same time. Flies lay eggs for approximately 15 days and begin to die around day 20.
  8. Feed the flies every day with agar-fruit juice plates topped with a spoonful of rehydrated baker´s yeast containing 200 µL of acetic acid or vinegar.
  9. To establish the next generation of flies, collect several plates of 0–12 h old embryos. Place the embryos in 22 cm × 17 cm × 10 cm plastic boxes containing fly food to a depth of 3 cm. Ensure that the boxes have a hole sealed with mesh, to allow air circulation and prevent larvae from escaping (Figure 4). Add dry active yeast on top of the food and allow the embryos to grow at 25 °C.
  10. After 3 days, place folded 5 cm tall paper into the boxes so that third-instar larvae can climb up and become pupae.
  11. After 10 days, place the boxes containing newly hatched adult flies into the cage. Allow the flies to exit the boxes and mate in the cage for 2 days.
  12. To obtain embryos of a specific age for lysate preparation, open the cage carefully and place agar-juice plates inside the cage for the required egg-laying period.
  13. Remove the plates and collect the eggs using a pile of sieves.
    NOTE: For 0–12 h-old embryo lysates, place two agar-juice plates into the cage at 8:00 a.m. and remove them at 8:00 p.m. for embryo collection.
    1. Collect embryos for lysate preparation between days 3 and 10 after adult flies hatch.
    2. In parallel, collect embryos on days 2–4 and establish a new generation of flies in food-containing boxes as described above.

4) Preparation of translation lysates from 0–12 h-old Drosophila embryos

NOTE: This protocol does not include micrococcal nuclease treatment. This step was omitted to avoid potential degradation of the exogenous reporter mRNA if nuclease inactivation is incomplete. Background translation should be monitored in each experiment using a no-mRNA control.
NOTE: This method is derived from previously described protocols7,12,14,15,16,17,22.

  1. Prepare embryo lysis buffer as described in Table 2 and store it at -20 °C in 1 mL aliquots.
    NOTE: Thaw each aliquot up to 2x for immediate use.
  2. Collect embryos using a sieve.
    NOTE: The standard protocol uses 0–12 h-old embryos, but other embryo ages may also be suitable for lysate preparation.
  3. Wash embryos with tap water extensively and then with embryo buffer containing 0.9% NaCl + 0.03% Triton X-100.
    NOTE: Embryos can be stored at 4 °C for up to 24 h before processing.
  4. Dechorionate embryos for 5 min in 50% embryo buffer and 50% house bleach (sodium hypochlorite [NaOCl] 4–6% and sodium hydroxide [NaOH] 0.02–0.1%).
    CAUTION: Use gloves and transparent plastic goggles when handling bleach.
  5. Wash extensively with tap water. Dry embryos with paper and weigh them.
  6. Transfer packed embryos to a glass tissue grinder homogenizer on ice. Add 1 mL of embryo lysis buffer per 1 g of embryos. Homogenize the embryos on ice with 20 strokes.
  7. Transfer the homogenate to 1.5 mL microcentrifuge tubes. Centrifuge at 15,900 × g for 20 min at 4 °C.
  8. Recover the interphase in 50 µL aliquots on ice. Freeze aliquots in liquid nitrogen and store at -80 °C.
    NOTE: The interphase is a cloudy liquid solution in between the upper and bottom phases. The upper phase is a layer of highly viscous, opaque-white lipids. The bottom phase is a solid pellet.
    CAUTION: Avoid contaminating the interphase solution from adjacent phases.
    CAUTION: Use gloves and transparent plastic goggles when handling liquid nitrogen.

5) In vitro translation reaction

  1. Set up the translation reactions in a 96-well plate or individual 0.2 mL PCR tubes on a PCR thermocycler. Prepare each translation reaction in a final volume of 17 µL, including 5 µL of embryo lysate and the remaining reaction components described in Table 2.
    NOTE: Embryo lysate represents 30% vol./vol. of the total volume of reaction.
  2. If recombinant proteins are added to the translation reaction, dialyze the protein against 20 mM HEPES-KOH pH 7.4. Select the dialysis cassette volume and molecular-weight cutoff according to the sample volume and protein size.
    NOTE: The addition of recombinant proteins is relevant to studying translational repressors or dose-dependent assays with initiation factors. Avoid adding PBS to recombinant proteins used in the translation reaction. Keep glycerol at or below 5% in the protein sample or translation reaction. If possible, avoid adding glycerol to the reaction.
  3. Denature the mRNA by heating to 65 °C for 2 min and quick chilling on ice for 2 min prior to the in vitro translation reaction.
  4. Carry out translation of each mRNA in triplicate. Set the reaction up as follows: 2.7 µL of reaction cocktail (Table 2) + 5 µL of embryo lysate + mRNA (30 ng) + H2O to 17 µL of final volume. As a base master mix, follow the Reaction cocktail for one reaction in Table 2 and scale to the number of reactions to be performed. Incubate the reaction at 25 °C for 60 min.
    NOTE: ATP and GTP are not added separately to the standard reaction described here. In our hands, supplementing the reaction with additional ATP/GTP did not improve FLuc output and reduced translation under the tested conditions. Therefore, the reaction is performed with the energy-regenerating system described in Table 2 without additional nucleotide supplementation.
  5. Program the thermocycler with an initial step of 5 min at 4 °C, followed by 60 min at 25 °C and a final step of 10 min at 4 °C.
  6. Stop the reaction by adding 20 µL of cold 1× passive lysis buffer. Place the reaction on ice for an additional 10 min.
  7. Store the samples at -20 °C or proceed to luciferase quantitation.
    NOTE: The protocol can be paused here.

6) Quantitation of synthesized luciferase

  1. Bring the reactions to room temperature for 20 min before quantifying FLuc activity.
  2. Bring 10 µL of each translation reaction and 50 µL of luciferase substrate (for at least 2 h before use) to room temperature.
  3. Add the luciferase substrate to the translation reaction. Record relative light units using an automatic or manual luminometer. Program the luminometer to take 10 readings spaced 1 s apart.

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Results

We performed translation reactions to analyze the efficiency variability among six independent lysate preparations (i.e., biological repetitions) using 0–12-h-old embryos. Each bar represents a technical triplicate (Figure 5). Similar translational efficiency was observed, although with some variability. We also compared translation efficiency among different age embryos. Thus, we tested lysates from 0–2-h-old, 0–12-h-old, and 0–18-h-old embryos, performing biological repetitions, that is, u...

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Discussion

Drosophila lysates for cell-free in vitro translation can yield reproducible results. Embryos at different ages and variations in buffer composition have been used to investigate a wide range of fundamental phenomena in protein synthesis and its machinery (Table 1). Here, we described a standard method suitable for embryos of any age for performing studies in a precise, quantitative manner (Table 1). This protocol is potentially useful for studying the function...

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Disclosures

The authors have no conflicts of interest to declare.

Acknowledgements

N.B-Á and G.H. were supported by the intramural funding program of National Institute of Cancer (Instituto Nacional de Cancerología, INCan), Mexico; D.E.V. is a posdoctoral fellow of the Secretaría de Ciencia, Humanidades, Tecnología e Innovación (SECIHTI) Program “Estancias posdoctorales por México para la Formación y Consolidación de las y los Investigadores por México, 2022”. We thank Fannis Missirlis and Bety Osorio (CINVESTAV, Mexico) for flies.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Aluminum foil sheetReynoldsN/AStandard household aluminum foil; any equivalent commercial brand acceptable.
Amino acids mixturePromegaL4461N/A
Apple cider vinegarHeinzN/ACommercial apple cider vinegar (5% acidity); any equivalent brand acceptable.
Apple or grape fruit juiceJumexN/ACommercial grape juice; or any equivalent commercial brand.
Bacteriological agar Merck/Sigma-Aldrich9002-18-0N/A
Boric acid (H3BO3)Merck/Sigma-AldrichB6768BioReagent, Molecular Biology grade, ≥99.5%, powder; used for preparation of TBE buffer.
Cap: Anti reverse cap (ARCA): 3´-O-Me-m7G(5´)ppp(5´)G RNA cap structureBioLabsS1411SN/A
Cap: m7G(5')ppp(5')G RNA cap structure analogNew England BiolabsS1404SN/A
Cap: Non-functional cap analogue G(5´)ppp(5´)ANew England BiolabsS1406N/A
Centrifuge Tube (15 mL)Corning 430053N/A
Complete, EDTA-free protease inhibitor cocktailRoche11836170001N/A
Creatine phosphokinase from rabbit muscleMerck/Sigma-AldrichC-3755N/A
Creatine phosphoric acid, disodium saltMerck1120A47950N/A
Dehydrated baker´s yeastFleischmann'sN/ACommercial active dry yeast; any equivalent brand acceptable.
Diethyl pyrocarbonate Merck/Sigma-AldrichD575897%(NNR)
DNA Clean & Concentrator-5Zymo ResearchD4004N/A
Drosophila melanogaster wild-type strain Oregon-RBloomington Drosophila Stock Center, Indiana, USAhttps://bdsc.indiana.edu/index.html ; mutant strains of Drosophila melanogaster, as well as wild-type reference strains of other Drosophila species, such as virilis, pseudoscura, grinshawi, hydei, etc. are also provided by this Stock Center.
Ethylenediaminetetraacetic acid (EDTA)Merck/Sigma-AldrichE9884ACS reagent, 99.4-100.6%, powder; used for preparation of TBE buffer.
HEPESSigmaSLBH4583V99.5%(titration)
Hydrogen peroxide (H2O2)EquateN/ACommercial (~2.5-3.5% hydrogen peroxide solution; any equivalent brand acceptable.
In vitro transcription mMessage mMachine T7, T3, or SP6 KitThermo Fisher ScientificAM1344 and AM1348N/A
KOAc, also termed KCH3COOMerck/Sigma-AldrichSeveralN/A
Lb broth (lennox)Merck/Sigma-AldrichL30221kg 
Luciferase assay systemPromega E1501N/A
MAXIscript T7, T3, or SP6 in vitro transcription KitInvitrogenAM1316 and AM1312N/A
Mg(OAc)2, also termed Mg(CH3COO)2Merck/Sigma-AldrichSeveralN/A
NaCl (sodium chloride)J.T Baker B17W53ACS Reagent
OligonucleotidesAny companyN/ASequence-specific.
Pasive lysis 5X bufferPromegaE1941N/A
Pipet TipsAxygenT-200-Y200 microliters yellow tips.
Pipet TipsAxygenT-1000-B1ml blue tips.
Pipet Tips AxygenT-3000.5-10 microliters clear tips.
Propionic AcidMerck/Sigma-Aldrich79-09-04ACS reagent grade, ≥99.5%, liquid.
PYREX® 5 mL Glass Pestle Tissue Grinder  PYREX®  7724-5N/A
QIAprep spin plasmid miniprep kitQiagen27106N/A
QIAquick gel extraction kitQiagen28704N/A
Rabbit reticulocyte lysate (RRL)Promega L4960N/A
Renilla luciferase (RLuc) assay systemPromega E2820N/A
RNasin ribonuclease inhibitorPromegaN2111AN/A
RNeasy minielute RNA clean up kitQiagen74204N/A
Slide-A-Lyzer Dialysis CassettesThermo Scientific87735 (However, it depends on the molecular size cutoff).N/A
Sodium hypochlorite (bleach)CloroxN/ACommercial bleach (~5-6% NaOCl); any equivalent commercial brand with similar concentration acceptable.
SpermidineMerck85558N/A
Stainless steel sieveN/AN/AAny commercial supplier.
Taq plus precision DNA polymeraseStratagene600211-51N/A
Transfer ribonucleic acid (tRNA) from bovine liver type XIMerck/Sigma-Aldrich9014-25-9 or R4752N/A
Tris (Tris base)IBI ScientificIB70145Molecular biology grade powder; used for preparation of TBE buffer.
Triton X-100SigmaSLBX9437Laboratory Grade
UltraPure AgaroseInvitrogen16500500N/A
Unflavored gelatin powderKnoxN/ACommercial unflavored gelatin powder; any equivalent brand acceptable.
Unrefined cane sugar (piloncillo)N/AN/ACommercial Mexican unrefined whole cane sugar; any equivalent brand acceptable.
Vector: pBluescript SK or KS (+/-)Stratagene200301N/A
Vector: pGEM-T or pGEM-T Easy. PromegaA3600 and A1360.N/A
Vector: pTZ57 R/T vector of the InsTAclone PCR Cloning kit ThermoFisher ScientificK1213 and K1214N/A
Wheat germ extract Promega L4380N/A
XhoI restriction enzyme New England BiolabsR0146N/A
ZymoPURE Plasmid Miniprep KitZymo ResearchD4212-BN/A
Equipment
Name of EquipmentCompanyCatalog NumberComments/Description
Accuris Analytical BalanceAccuris InstrumentsW3100A-120N/A
Epoch Microplate SpectrophotometerBioTek InstrumentsBT7310043N/A
Finnpipette F2Thermo ScientificLH63501100-1000 microliter pipette
Finnpipette F2Thermo Scientific464208020-200 microliter pipette
Finnpipette F2Thermo Scientific46420502-20 microliter pipette
Finnpipette F2Thermo Scientific46420100.2-2 microliter pipette
Finnpipette F2Thermo Scientific46420301-10 microliter pipette
GloMax 20/20 LuminometerPromegaE5311N/A
Heratherm Incubator IMC 18Thermo Scientific50126464N/A
MaxQ 4450 Benchtop Orbital ShakerThermo ScientificSHKE4450N/A
MultiGene OptiMax Thermal CyclerLabnet InternationalTC9610115V Model
Prism R Refrigerated MicroCentrifugeLabnet InternationalC2500-RWith 24 place rotor, 115V
UVP MultiDoc-It Imaging SystemUVPUVP97019501N/A

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Cell-Free TranslationIn Vitro TranslationProtein SynthesisTranslation MachinerySynthetic mRNA TemplatesTranslational ControlDrosophila EmbryosTranslation Protocol
Cell-Free In Vitro mRNA Translation In Drosophila Lysates
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