ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

N.B-&#193; and G.H. were supported by the intramural funding program of National Institute of Cancer (Instituto Nacional de Cancerolog&#237;a, INCan), Mexico; D.E.V. is a posdoctoral fellow of the Secretar&#237;a de Ciencia, Humanidades, Tecnolog&#237;a e Innovaci&#243;n (SECIHTI) Program &#8220;Estancias posdoctorales por M&#233;xico para la Formaci&#243;n y Consolidaci&#243;n de las y los Investigadores por M&#233;xico, 2022&#8221;. We thank Fannis Missirlis and Bety Osorio (CINVESTAV, Mexico) for flies.

1) Template plasmid construction for in vitro transcription
<ol>
	<li>Select plasmids containing a 5&#180; untranslated region (UTR), a reporter cistron, a 3&#180; 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&#39; overhang at the 3&#39; end of the template. 3&#39; overhangs can result in aberrant transcription products. If an enzyme with a 3&#39; overhang is used, the fragment should be blunted before use.</li>
	<li>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.</li>
</ol>
2) In vitro transcription of capped and polyadenylated mRNAs
<ol>
	<li>Linearize each plasmid construct using the unique restriction site downstream of the poly(A) tail (here, KpnI).</li>
	<li>Resolve the linearized DNA by 1%-agarose gel electrophoresis run at 100 V for 1 h.</li>
	<li>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&#8212;if the DNA is completely cut, then a column purification or phenol-extraction/ETOH precipitation is sufficient.</li>
	<li>Transcribe the purified linearized DNA using an in vitro transcription kit according to the manufacturer&#8217;s instructions. Include a 5&#180; cap analog in the transcription reaction to stabilize the RNA and stimulate translation. 
	NOTE: A functional cap analog, such as m7G(5&#8242;)ppp(5&#8242;)G RNA, supports transcript stabilization and translation stimulation. A non-functional cap analog, such as G(5&#8242;)ppp(5&#8242;)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.</li>
	<li>Treat all the transcription reactions with RNase-free DNase to remove the DNA template after transcription according to the manufacturer&#8217;s instructions. All in vitro transcription kits provide the RNase-free DNase.</li>
	<li>Purify the transcript by lithium chloride precipitation or column-based RNA cleanup according to the manufacturer&#8217;s instructions. 
	NOTE: All in vitro transcription kits provide the lithium chloride reagents.</li>
	<li>Quantify by spectroscopy the purified transcript and assess mRNA integrity by 1%-agarose gel electrophoresis run at 100 V for 1 h.</li>
	<li>Aliquot the mRNA and store at -70 &#176;C until use.</li>
</ol>
3) Routine maintenance of a large-scale Drosophila culture
<ol>
	<li>Use the reference Drosophila melanogaster wild-type strain Oregon-R.</li>
	<li>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.</li>
	<li>Set a cubic or cylindrical acrylic cage approximately 1 m long and 50 cm in diameter.</li>
	<li>Make an approximately 30 cm diameter hole in the upper part of the cage and seal it with mesh (Figure 3).</li>
	<li>Place wet paper on top of the mesh to provide water for the flies, and replace the wet paper every day (Figure 3).</li>
	<li>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.</li>
	<li>Amplify the fly stock before embryo collection, and place ~1,000 young flies inside the cage. Maintain the flies&#39; cage at 22&#8211;25 &#176;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.</li>
	<li>Feed the flies every day with agar-fruit juice plates topped with a spoonful of rehydrated baker&#180;s yeast containing 200 &#181;L of acetic acid or vinegar.</li>
	<li>To establish the next generation of flies, collect several plates of 0&#8211;12 h old embryos. Place the embryos in 22 cm &#215; 17 cm &#215; 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 &#176;C.</li>
	<li>After 3 days, place folded 5 cm tall paper into the boxes so that third-instar larvae can climb up and become pupae.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Remove the plates and collect the eggs using a pile of sieves. 
	NOTE: For 0&#8211;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.
	<ol>
		<li>Collect embryos for lysate preparation between days 3 and 10 after adult flies hatch.</li>
		<li>In parallel, collect embryos on days 2&#8211;4 and establish a new generation of flies in food-containing boxes as described above.</li>
	</ol>
	</li>
</ol>
4) Preparation of translation lysates from 0&#8211;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.
<ol>
	<li>Prepare embryo lysis buffer as described in Table 2 and store it at -20 &#176;C in 1 mL aliquots. 
	NOTE: Thaw each aliquot up to 2x for immediate use.</li>
	<li>Collect embryos using a sieve. 
	NOTE: The standard protocol uses 0&#8211;12 h-old embryos, but other embryo ages may also be suitable for lysate preparation.</li>
	<li>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 &#176;C for up to 24 h before processing.</li>
	<li>Dechorionate embryos for 5 min in 50% embryo buffer and 50% house bleach (sodium hypochlorite [NaOCl] 4&#8211;6% and sodium hydroxide [NaOH] 0.02&#8211;0.1%). 
	CAUTION: Use gloves and transparent plastic goggles when handling bleach.</li>
	<li>Wash extensively with tap water. Dry embryos with paper and weigh them.</li>
	<li>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.</li>
	<li>Transfer the homogenate to 1.5 mL microcentrifuge tubes. Centrifuge at 15,900 &#215; g for 20 min at 4 &#176;C.</li>
	<li>Recover the interphase in 50 &#181;L aliquots on ice. Freeze aliquots in liquid nitrogen and store at -80 &#176;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.</li>
</ol>
5) In vitro translation reaction
<ol>
	<li>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 &#181;L, including 5 &#181;L 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.</li>
	<li>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.</li>
	<li>Denature the mRNA by heating to 65 &#176;C for 2 min and quick chilling on ice for 2 min prior to the in vitro translation reaction.</li>
	<li>Carry out translation of each mRNA in triplicate. Set the reaction up as follows: 2.7 &#181;L of reaction cocktail (Table 2) + 5 &#181;L of embryo lysate + mRNA (30 ng) + H2O to 17 &#181;L 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 &#176;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.</li>
	<li>Program the thermocycler with an initial step of 5 min at 4 &#176;C, followed by 60 min at 25 &#176;C and a final step of 10 min at 4 &#176;C.</li>
	<li>Stop the reaction by adding 20 &#181;L of cold 1&#215; passive lysis buffer. Place the reaction on ice for an additional 10 min.</li>
	<li>Store the samples at -20 &#176;C or proceed to luciferase quantitation. 
	NOTE: The protocol can be paused here.</li>
</ol>
6) Quantitation of synthesized luciferase
<ol>
	<li>Bring the reactions to room temperature for 20 min before quantifying FLuc activity.</li>
	<li>Bring 10 &#181;L of each translation reaction and 50 &#181;L of luciferase substrate (for at least 2 h before use) to room temperature.</li>
	<li>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.</li>
</ol>

<ol>
	<li>Marygold SJ, Attrill H, Lasko P. The translation factors of Drosophila melanogaster. Fly (Austin). 2017;11(1):65-74.</li>
	<li>Hern&#225;ndez G, Vazquez-Pianzola P. eIF4E as a molecular wildcard in metazoans RNA metabolism. Biol Rev Camb Philos Soc. 2023;98(6):2284-306.</li>
	<li>Kong J, Lasko P. Translational control in cellular and developmental processes. Nat Rev Genet. 2012;13(6):383-94.</li>
	<li>Bayer LV, Milano SN, Bratu DP. The mRNA dynamics underpinning translational control mechanisms of Drosophila melanogaster oogenesis. Biochem Soc Trans. 2024;52(5):2087-99.</li>
	<li>Filipowicz W, et al. A protein binding the methylated 5&#8242;-terminal sequence, m7GpppN, of eukaryotic messenger RNA. Proc Natl Acad Sci U S A. 1976;73(5):1559-63.</li>
	<li>Sondermeijer PJA, Lubsen NH. Heat-shock peptides in Drosophila hydei and their synthesis in vitro. Eur J Biochem. 1978;88(2):331-9.</li>
	<li>Hern&#225;ndez G, Vazquez-Pianzola P, Sierra JM, Rivera-Pomar R. Internal ribosome entry site drives cap-independent translation of reaper and heat shock protein 70 mRNAs in Drosophila embryos. RNA. 2004;10(11):1783-97.</li>
	<li>Zapata JM, Mart&#237;nez MA, Sierra JM. Purification and characterization of eukaryotic polypeptide chain initiation factor 4F from Drosophila melanogaster embryos. J Biol Chem. 1994;269(27):18047-52.</li>
	<li>Maroto FG, Sierra JM. Purification and characterization of mRNA cap-binding protein from Drosophila melanogaster embryos. Mol Cell Biol. 1989;9(5):2181-90.</li>
	<li>Andrews S, Snowflack DR, Clark IE, Gavis ER. Multiple mechanisms collaborate to repress nanos translation in the Drosophila ovary and embryo. RNA. 2011;17(5):967-77.</li>
	<li>Smibert CA, et al. Smaug, a novel and conserved protein, contributes to repression of nanos mRNA translation in vitro. RNA. 1999;5(12):1535-47.</li>
	<li>Gebauer F, et al. Translational control of dosage compensation in Drosophila by Sex-lethal: cooperative silencing via the 5&#8242; and 3&#8242; UTRs of msl-2 mRNA is independent of the poly(A) tail. EMBO J. 1999;18(21):6146-54.</li>
	<li>Pamudurti NR, et al. Translation of circRNAs. Mol Cell. 2017;66(1):9-21.e7.</li>
	<li>V&#225;zquez-Pianzola P, Hern&#225;ndez G, Suter B, Rivera-Pomar R. Different modes of translation for hid, grim and sickle mRNAs in Drosophila. Cell Death Differ. 2007;14(2):286-95.</li>
	<li>Hern&#225;ndez G, et al. Two functionally redundant isoforms of Drosophila eIF4B are involved in cap-dependent translation, cell survival and proliferation. Eur J Biochem. 2004;271(14):2923-36.</li>
	<li>Hern&#225;ndez G, et al. Functional analysis of the AUG initiator codon context reveals novel conserved sequences that disfavor mRNA translation in eukaryotes. Nucleic Acids Res. 2024;52(3):1064-79.</li>
	<li>Tuschl T, et al. Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev. 1999;13(24):3191-7.</li>
	<li>Scott MP, Storti RV, Pardue ML, Rich A. Cell-free protein synthesis in lysates of Drosophila melanogaster cells. Biochemistry. 1979;18(8):1588-94.</li>
	<li>Clark IE, Wyckoff D, Gavis ER. Synthesis of the posterior determinant Nanos is spatially restricted by a novel cotranslational regulatory mechanism. Curr Biol. 2000;10(20):1311-4.</li>
	<li>Jeske M, et al. Rapid ATP-dependent deadenylation of nanos mRNA in a cell-free system from Drosophila embryos. J Biol Chem. 2006;281(35):25124-33.</li>
	<li>Jeske M, Moritz B, Anders A, Wahle E. Smaug assembles an ATP-dependent stable complex repressing nanos mRNA translation at multiple levels. EMBO J. 2011;30(1):90-103.</li>
	<li>Castagnetti S, Hentze MW, Ephrussi A, Gebauer F. Control of oskar mRNA translation by Bruno in a novel cell-free system from Drosophila ovaries. Development. 2000;127(5):1063-8.</li>
	<li>Gebauer F, Hentze MW. Studying translational control in Drosophila cell-free systems. Methods Enzymol. 2007;429:23-33.</li>
	<li>Hern&#225;ndez G, et al. Mextli is a novel eukaryotic translation initiation factor 4E-binding protein that promotes translation in Drosophila melanogaster. Mol Cell Biol. 2013;33(15):2854-64.</li>
	<li>Lie YS, Macdonald PM. In vitro translation extracts prepared from Drosophila ovaries and embryos. Biochem Biophys Res Commun. 2000;270(2):473-81.</li>
	<li>Peng Y, Gavis ER. The Drosophila hnRNP F/H homolog Glorund recruits dFMRP to inhibit nanos translation elongation. Nucleic Acids Res. 2022;50(12):7067-83.</li>
	<li>Sanders MM, Triemer DF, Olsen AS. Regulation of protein synthesis in heat-shocked Drosophila cells: soluble factors control translation in vitro. J Biol Chem. 1986;261(5):2189-96.</li>
	<li>Storti RV, Scott MP, Rich A, Pardue ML. Translational control of protein synthesis in response to heat shock in Drosophila melanogaster cells. Cell. 1980;22(3):825-34.</li>
	<li>Kr&#252;ger C, Benecke BJ. In vitro translation of Drosophila heat-shock and non-heat-shock mRNAs in heterologous and homologous cell-free systems. Cell. 1981;23(2):595-603.</li>
	<li>Zhao F, Yu CH, Liu Y. Codon usage regulates protein structure and function by affecting translation elongation speed in Drosophila cells. Nucleic Acids Res. 2017;45(14):8484-92.</li>
	<li>Roy G, et al. The Drosophila poly(A) binding protein-interacting protein, dPaip2, is a novel effector of cell growth. Mol Cell Biol. 2004;24(3):1143-54.</li>
	<li>Wakiyama M, Yokoyama S. Posttranscriptional control of protein synthesis in Drosophila S2 cell-free system. Methods Mol Biol. 2014;1118:257-66.</li>
	<li>Spirin AS, Swartz JR. Cell-free protein synthesis systems: historical landmarks, classification, and general methods. In: Spirin AS, Swartz JR, editors. Cell-free protein synthesis. Weinheim: Wiley-VCH; 2008. p. 1-34.</li>
	<li>Gregorio NE, Levine MZ, Oza JP. A user&#8217;s guide to cell-free protein synthesis. Methods Protoc. 2019;2(1):24.</li>
	<li>Hern&#225;ndez G, Altmann M, Lasko P. Origins and evolution of the mechanisms regulating translation initiation in eukaryotes. Trends Biochem Sci. 2010;35(2):63-73.</li>
	<li>Hern&#225;ndez G, Proud C, Preiss T, Parsyan A. On the diversification of the translational apparatus across eukaryotes. Comp Funct Genomics. 2012;2012:256848.</li>
	<li>Lakovaara S. Malt as a culture medium for Drosophila species. Drosoph Inf Serv. 1969;44:128.</li>
</ol>

The authors have no conflicts of interest to declare.

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...

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.

<table><tbody><tr><td>Aluminum foil sheet</td><td>Reynolds</td><td>N/A</td><td>Standard household aluminum foil; any equivalent commercial brand acceptable.</td></tr><tr><td>Amino acids mixture</td><td>Promega</td><td>L4461</td><td>N/A</td></tr><tr><td>Apple cider vinegar</td><td>Heinz</td><td>N/A</td><td>Commercial apple cider vinegar (5% acidity); any equivalent brand acceptable.</td></tr><tr><td>Apple or grape fruit juice</td><td>Jumex</td><td>N/A</td><td>Commercial grape juice; or any equivalent commercial brand.</td></tr><tr><td>Bacteriological agar&amp;nbsp;</td><td>Merck/Sigma-Aldrich</td><td>9002-18-0</td><td>N/A</td></tr><tr><td>Boric acid (H&lt;sub&gt;3&lt;/sub&gt;BO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Merck/Sigma-Aldrich</td><td>B6768</td><td>BioReagent, Molecular Biology grade, &amp;ge;99.5%, powder; used for preparation of TBE buffer.</td></tr><tr><td>Cap: Anti reverse cap (ARCA): 3&amp;acute;-O-Me-m&lt;sup&gt;7&lt;/sup&gt;G(5&amp;acute;)ppp(5&amp;acute;)G RNA cap structure</td><td>BioLabs</td><td>S1411S</td><td>N/A</td></tr><tr><td>Cap: m&lt;sup&gt;7&lt;/sup&gt;G(5&#039;)ppp(5&#039;)G RNA cap structure analog</td><td>New England Biolabs</td><td>S1404S</td><td>N/A</td></tr><tr><td>Cap: Non-functional cap analogue G(5&amp;acute;)ppp(5&amp;acute;)A</td><td>New England Biolabs</td><td>S1406</td><td>N/A</td></tr><tr><td>Centrifuge Tube (15 mL)</td><td>Corning&amp;nbsp;</td><td>430053</td><td>N/A</td></tr><tr><td>Complete, EDTA-free protease inhibitor cocktail</td><td>Roche</td><td>11836170001</td><td>N/A</td></tr><tr><td>Creatine phosphokinase from rabbit muscle</td><td>Merck/Sigma-Aldrich</td><td>C-3755</td><td>N/A</td></tr><tr><td>Creatine phosphoric acid, disodium salt</td><td>Merck</td><td>1120A47950</td><td>N/A</td></tr><tr><td>Dehydrated baker&amp;acute;s yeast</td><td>Fleischmann&#039;s</td><td>N/A</td><td>Commercial active dry yeast; any equivalent brand acceptable.</td></tr><tr><td>Diethyl pyrocarbonate&amp;nbsp;</td><td>Merck/Sigma-Aldrich</td><td>D5758</td><td>97%(NNR)</td></tr><tr><td>DNA Clean &amp;amp; Concentrator-5</td><td>Zymo Research</td><td>D4004</td><td>N/A</td></tr><tr><td>Drosophila melanogaster wild-type strain Oregon-R</td><td>Bloomington Drosophila Stock Center, Indiana, USA</td><td></td><td>https://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.</td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Merck/Sigma-Aldrich</td><td>E9884</td><td>ACS reagent, 99.4-100.6%, powder; used for preparation of TBE buffer.</td></tr><tr><td>HEPES</td><td>Sigma</td><td>SLBH4583V</td><td>99.5%(titration)</td></tr><tr><td>Hydrogen peroxide (H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;)</td><td>Equate</td><td>N/A</td><td>Commercial (~2.5-3.5% hydrogen peroxide solution; any equivalent brand acceptable.</td></tr><tr><td>In vitro transcription mMessage mMachine T7, T3, or SP6 Kit</td><td>Thermo Fisher Scientific</td><td>AM1344 and AM1348</td><td>N/A</td></tr><tr><td>KOAc, also termed KCH&lt;sub&gt;3&lt;/sub&gt;COO</td><td>Merck/Sigma-Aldrich</td><td>Several</td><td>N/A</td></tr><tr><td>Lb broth (lennox)</td><td>Merck/Sigma-Aldrich</td><td>L3022</td><td>1kg&amp;nbsp;</td></tr><tr><td>Luciferase assay system</td><td>Promega&amp;nbsp;</td><td>E1501</td><td>N/A</td></tr><tr><td>MAXIscript T7, T3, or SP6 in vitro transcription Kit</td><td>Invitrogen</td><td>AM1316 and AM1312</td><td>N/A</td></tr><tr><td>Mg(OAc)&lt;sub&gt;2&lt;/sub&gt;, also termed Mg(CH&lt;sub&gt;3&lt;/sub&gt;COO)&lt;sub&gt;2&lt;/sub&gt;</td><td>Merck/Sigma-Aldrich</td><td>Several</td><td>N/A</td></tr><tr><td>NaCl (sodium chloride)</td><td>J.T Baker&amp;nbsp;</td><td>B17W53</td><td>ACS Reagent</td></tr><tr><td>Oligonucleotides</td><td>Any company</td><td>N/A</td><td>Sequence-specific.</td></tr><tr><td>Pasive lysis 5X buffer</td><td>Promega</td><td>E1941</td><td>N/A</td></tr><tr><td>Pipet Tips</td><td>Axygen</td><td>T-200-Y</td><td>200 microliters yellow tips.</td></tr><tr><td>Pipet Tips</td><td>Axygen</td><td>T-1000-B</td><td>1ml blue tips.</td></tr><tr><td>Pipet Tips&amp;nbsp;</td><td>Axygen</td><td>T-300</td><td>0.5-10 microliters clear tips.</td></tr><tr><td>Propionic Acid</td><td>Merck/Sigma-Aldrich</td><td>79-09-04</td><td>ACS reagent grade, &amp;ge;99.5%, liquid.</td></tr><tr><td>PYREX&amp;reg; 5 mL Glass Pestle Tissue Grinder</td><td>&amp;nbsp; PYREX&amp;reg;&amp;nbsp;&amp;nbsp;</td><td>7724-5</td><td>N/A</td></tr><tr><td>QIAprep spin plasmid miniprep kit</td><td>Qiagen</td><td>27106</td><td>N/A</td></tr><tr><td>QIAquick gel extraction kit</td><td>Qiagen</td><td>28704</td><td>N/A</td></tr><tr><td>Rabbit reticulocyte lysate (RRL)</td><td>Promega&amp;nbsp;</td><td>L4960</td><td>N/A</td></tr><tr><td>Renilla luciferase (RLuc) assay system</td><td>Promega&amp;nbsp;</td><td>E2820</td><td>N/A</td></tr><tr><td>RNasin ribonuclease inhibitor</td><td>Promega</td><td>N2111A</td><td>N/A</td></tr><tr><td>RNeasy minielute RNA clean up kit</td><td>Qiagen</td><td>74204</td><td>N/A</td></tr><tr><td>Slide-A-Lyzer Dialysis Cassettes</td><td>Thermo Scientific</td><td>87735 (However, it depends on the molecular size cutoff).</td><td>N/A</td></tr><tr><td>Sodium hypochlorite (bleach)</td><td>Clorox</td><td>N/A</td><td>Commercial bleach (~5-6% NaOCl); any equivalent commercial brand with similar concentration acceptable.</td></tr><tr><td>Spermidine</td><td>Merck</td><td>85558</td><td>N/A</td></tr><tr><td>Stainless steel sieve</td><td>N/A</td><td>N/A</td><td>Any commercial supplier.</td></tr><tr><td>Taq plus precision DNA polymerase</td><td>Stratagene</td><td>600211-51</td><td>N/A</td></tr><tr><td>Transfer ribonucleic acid (tRNA) from bovine liver type XI</td><td>Merck/Sigma-Aldrich</td><td>9014-25-9 or R4752</td><td>N/A</td></tr><tr><td>Tris (Tris base)</td><td>IBI Scientific</td><td>IB70145</td><td>Molecular biology grade powder; used for preparation of TBE buffer.</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>SLBX9437</td><td>Laboratory Grade</td></tr><tr><td>UltraPure Agarose</td><td>Invitrogen</td><td>16500500</td><td>N/A</td></tr><tr><td>Unflavored gelatin powder</td><td>Knox</td><td>N/A</td><td>Commercial unflavored gelatin powder; any equivalent brand acceptable.</td></tr><tr><td>Unrefined cane sugar (piloncillo)</td><td>N/A</td><td>N/A</td><td>Commercial Mexican unrefined whole cane sugar; any equivalent brand acceptable.</td></tr><tr><td>Vector: pBluescript SK or KS (+/-)</td><td>Stratagene</td><td>200301</td><td>N/A</td></tr><tr><td>Vector: pGEM-T or pGEM-T Easy.&amp;nbsp;</td><td>Promega</td><td>A3600 and A1360.</td><td>N/A</td></tr><tr><td>Vector: pTZ57 R/T vector of the InsTAclone PCR Cloning kit&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>K1213 and K1214</td><td>N/A</td></tr><tr><td>Wheat germ extract&amp;nbsp;</td><td>Promega&amp;nbsp;</td><td>L4380</td><td>N/A</td></tr><tr><td>XhoI restriction enzyme&amp;nbsp;</td><td>New England Biolabs</td><td>R0146</td><td>N/A</td></tr><tr><td>ZymoPURE Plasmid Miniprep Kit</td><td>Zymo Research</td><td>D4212-B</td><td>N/A</td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>Name of Equipment</td><td>Company</td><td>Catalog Number</td><td>Comments/Description</td></tr><tr><td>Accuris Analytical Balance</td><td>Accuris Instruments</td><td>W3100A-120</td><td>N/A</td></tr><tr><td>Epoch Microplate Spectrophotometer</td><td>BioTek Instruments</td><td>BT7310043</td><td>N/A</td></tr><tr><td>Finnpipette F2</td><td>Thermo Scientific</td><td>LH63501</td><td>100-1000 microliter pipette</td></tr><tr><td>Finnpipette F2</td><td>Thermo Scientific</td><td>4642080</td><td>20-200 microliter pipette</td></tr><tr><td>Finnpipette F2</td><td>Thermo Scientific</td><td>4642050</td><td>2-20 microliter pipette</td></tr><tr><td>Finnpipette F2</td><td>Thermo Scientific</td><td>4642010</td><td>0.2-2 microliter pipette</td></tr><tr><td>Finnpipette F2</td><td>Thermo Scientific</td><td>4642030</td><td>1-10 microliter pipette</td></tr><tr><td>GloMax 20/20 Luminometer</td><td>Promega</td><td>E5311</td><td>N/A</td></tr><tr><td>Heratherm Incubator IMC 18</td><td>Thermo Scientific</td><td>50126464</td><td>N/A</td></tr><tr><td>MaxQ 4450 Benchtop Orbital Shaker</td><td>Thermo Scientific</td><td>SHKE4450</td><td>N/A</td></tr><tr><td>MultiGene OptiMax Thermal Cycler</td><td>Labnet International</td><td>TC9610</td><td>115V Model</td></tr><tr><td>Prism R Refrigerated MicroCentrifuge</td><td>Labnet International</td><td>C2500-R</td><td>With 24 place rotor, 115V</td></tr><tr><td>UVP MultiDoc-It Imaging System</td><td>UVP</td><td>UVP97019501</td><td>N/A</td></tr></tbody></table>

cell-free in vitro mrna translation in drosophila lysates

Cell-Free In Vitro mRNA Translation In Drosophila Lysates

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&#8212;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.

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...

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

Cell-free mRNA in vitro translation has played a crucial role in the understanding of the protein synthesis process during gene expression across ...

Our research employs a drosophila cell-free translation system to study protein synthesis and translational revelation across various stages of insect development. This protocol accurately recapitulates in vivo translation events specific to insect development, unlike the lysates from distant metazoans. To begin, open the Benchling cloning software.Select a suitable plasmid to insert the construct containing a five prime untranslated region, a firefly luciferase, or Fluc reporter cistron, a three prime untranslated region, and a polyadenylate or poly(A)tail downstream of a T7 promoter. Use the unique restriction site of KPN1 immediately downstream of the poly(A)tail to linearize each plasmid construct before transcription. Verify the clean, well-resolved peaks of the constructs obtained by sequencing before using them as templates for in vitro transcription.Resolve the linearized DNA by 1%agaros gel electrophoresis at 100 volts for one hour. Gel extract the linearized DNA. Place it in a microtube and add agaros digestion buffer or ADB for DNA purification.Incubate the tube in a thermoshaker. Then transfer the contents to a column and centrifuge to recover the linearized DNA. Use an in vitro transcription kit to transcribe the linearized DNA according to the manufacturer's instructions.Load the thermocycler and select the appropriate program. Treat all transcription reactions with RNase-free DNase to remove the DNA template after transcription. Purify the transcript using lithium chloride precipitation following the manufacturer's instructions.Quantify the purified transcript by spectroscopy. Assess messenger RNA or MRNA integrity by 1%agaros gel electrophoresis at 100 volts for one hour. Aliquot the mRNA and store it at minus 80 degrees Celsius until use.For culturing the reference, drosophila melanogaster wild-type strain Oregon-R, set up a cylindrical acrylic cage approximately one meter long and 50 centimeters in diameter with an opening of approximately 30 centimeters in diameter in the upper part. Place a mesh to cover the upper opening and seal it with tape on all four sides. Place wet paper on top of the mesh to provide water for the flies, replacing it every day.Keep the front part of the cage open and covered with fabric for access. From the amplified fly stock, place approximately 1, 000 young flies inside the cage, maintained at 22 to 25 degrees Celsius. To prepare the feed, top the egger fruit juice plates with a spoonful of rehydrated baker's yeast, then add 200 microliters of vinegar uniformly throughout the plate.Carefully insert the feed plate through the front opening of the cage and seal the fabric to contain the flies. To collect the embryos, remove the plates from the cage. Prepare embryo lysis buffer and store one milliliter aliquots at minus 80 degrees Celsius.Use a sieve to collect the embryos, then wash them extensively with tap water, followed by embryo buffer containing 0.9%sodium chloride and 0.03%Triton X100. Dechorionate the embryos for five minutes in a solution containing 50%embryo buffer and 50%household bleach. Wash the embryos extensively with tap water, then dry them with paper and weigh them.Transfer the packed embryos to a glass tissue grinder homogenizer on ice. Add one milliliter of embryolysis buffer per one gram of embryos. Homogenize the embryos on ice with 20 strokes.Transfer the homogenate to 1.5 milliliter microcentrifuge tubes. Centrifuge the homogenate at 15, 900 G for 20 minutes at four degrees Celsius. Recover the interface in 50 microliter aliquots on ice.Store the aliquots at minus 80 degrees Celsius immediately. To set up the translation reactions, first denature the mRNA sample at 65 degrees Celsius for two minutes, followed by chilling at four degrees for two minutes. Then set up the reactions in triplicate for each mRNA in individual 0.2 milliliter PCR tubes by adding 2.7 microliters of reaction mix, five microliters of the embryo lysate, 30 nanograms of the denatured RNA, and water to reach a final volume of 17 microliters.Place the reaction tubes in the thermocycler, then program the thermocycler with an initial step of five minutes at four degrees Celsius, followed by 60 minutes at 25 degrees Celsius, and a final 10 minutes at four degrees Celsius. To stop the reaction, add 20 microliters of cold 1X passive lysis buffer and mix thoroughly. Place the reactions at room temperature and proceed directly to luciferase quantitation.Add 50 microliters of luciferase substrate to 10 microliters of translation reaction. Record relative light units, or RLU, using an automatic or manual luminometer programmed to collect three readings at three second intervals. Analysis of efficiency variability among six independent lysate preparations using zero to 12-hour old embryos showed similar translational efficiency, although some variability was present.Lysate number six was significantly different from all other lysates. Comparison of translation efficiency among different age embryos showed that lysates from zero to two-hour old embryos produced the lowest firefly luciferase activity, whereas lysates from zero to 18-hour old embryos produced the highest activity. Comparison of the translation efficiency of 30%or 70%lysate volumes of drosophila zero to 18-hour old embryo with commercial rabbit reticulocyte and wheat germ lysates showed that rabbit reticulocyte lysates were the most efficient translation system and drosophila embryo lysates were the least efficient.This protocol allows researchers to study mRNA translation and translational control mechanisms using drosophila embryolysates. The critical challenges are maintaining mRNA integrity and minimizing lysate variability through consistent embryo aging and homogenization. Future studies can extend this protocol to other insect species for evolutionary comparisons and to investigate novel translational dynamics during embryogenesis.

cell-free-in-vitro-mrna-translation-in-drosophila-lysates

Research

JoVE Journal

Biochemistry

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

Purification of Transcripts and Metabolites from Drosophila Heads

The aim of this publication is to visualize and discuss the operative steps of an Enhanced Northern Blot protocol on RNA extracted from Drosophila melanogaster embryos, cells, and tissues. This protocol is particularly useful for the efficient detection of small RNA species.

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Xenopus laevis as a Model to Identify Translation Impairment

Translating Ribosome Affinity Purification (TRAP) is able to capture cell-type-specific translation of mRNA. Here we report the first TRAP protocol dedicated to isolation of mRNA in rare cell populations of Drosophila embryos.

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

The goal of this protocol is to provide a straightforward and inexpensive approach to collecting Drosophila embryos at medium scale (0.5-1 g) and preparing protein extracts that can be used in downstream proteomic applications, such as affinity purification-mass spectrometry (AP-MS).

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

A tandem RNA isolation procedure (TRIP) for recovery of endogenously formed mRNA-protein complexes is described. Specifically, RNA-protein complexes are crosslinked in vivo, polyadenylated RNAs are isolated from extracts with oligo(dT) beads, and particular mRNAs are captured with modified RNA antisense oligonucleotides. Proteins bound to mRNAs are detected by immunoblot analysis.

An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

This article outlines a detailed protocol for using the RNA-targeting Cas13D enzyme (RfxCas13D) in flies.

Ubiquitous and Tissue-specific RNA Targeting in Drosophila Melanogaster using CRISPR/CasRx

This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and presents an optimized protocol for embryo microinjection, a crucial step for creating transgenic flies.

Embryo Microinjection for Transgenesis in Drosophila

Instituto Nacional de Cancerología (INCan; National Institute of Cancer).  Here, we provide details on how to prepare lysates and perform in vitro translation using Drosophila embryos.

Cell-Free In Vitro mRNA Translation In Drosophila Lysates

Explore More Videos