The work was supported by the Scientific Research Fund for High-Level Talents, University of South China.

1. Preparation of plasmids
<ol>
	<li>Isolate plasmids from 4 mL overnight bacterial cultures using a plasmid miniprep kit. Elute with 40 &#181;L of elution buffer. 
	NOTE: Isolation of plasmids using a plasmid midi-prep kit is not necessary. A plasmid miniprep kit can fully meet the experimental requirements for embryo microinjection. In this protocol, the prepared UAS-cDNA/ORF plasmids contain ten copies of UAS, an Hsp70 minimal promoter, an attB site, a mini-white gene, and a gene of interest.</li>
	<li>Determine the plasmid DNA concentration using a spectrophotometer and store it at -80 &#176;C freezer. 
	NOTE: Plasmid concentration is not critical for successful embryo microinjection, as plasmid concentrations ranging from 20 ng/&#181;L to 1683 ng/&#181;L work well for transgenesis in Drosophila (Supplementary Table 1).</li>
	<li>Just before microinjection, thaw plasmids and centrifuge at 13000 x g for 5 min.</li>
	<li>Transfer 10 &#181;L of each plasmid from the upper layer of the liquid into a new 1.5 mL tube.</li>
</ol>
2. Pulling needles
<ol>
	<li>Turn on a needle puller with selected settings (Heat: 500, Pull: 50, Vel: 60, Delay: 90, Pressure: 200, Ramp Test: 510).</li>
	<li>Insert a borosilicate glass capillary (outer diameter 1.0 mm, inner diameter 0.75 mm, 10 cm length) into the needle puller and close the lid.</li>
	<li>Press the Pull button. Each glass capillary makes two needles.</li>
</ol>
3. Grinding needles (Figure 1)
<ol>
	<li>Use a needle grinder mounted with an appropriate abrasive plate (for 0.7-2.0 &#181;m tip sizes) of a needle grinder.</li>
	<li>Wet the cloth (reference wire) with grinding solution (100 mL of distilled water with 0.9 g of NaCl, 1 mL of wetting agent) and fix the cloth.</li>
	<li>Turn on the needle grinder and keep the grinding plate rotating.</li>
	<li>Connect a needle to a foot air pump with a pressure gauge via a silicone capillary tube, and then mount the needle onto a needle holder of the needle grinder. Adjust the angle of the needle relative to the grinding plate to 35&#176; (Figure 1A, B).</li>
	<li>Adjust the position of the needle tip just above the grinding plate surface by using a coarse control knob under the microscope (Figure 1C). 
	NOTE: The pulled needles obtained in section 2 have a 6-8 mm taper and a less than 1 &#181;m tip (Figure 1D), as is described in the user manual of the puller.</li>
	<li>Keep the inner pressure of the needle at 40 psi using the foot air pump. Adjust the position of the needle tip by using a fine control knob under the microscope, and make the tip of the needle touch the grinding plate.</li>
	<li>Grind for 3-5&#160;s. When tiny air bubbles come out of the needle tip, unload the needle (Figure 1E). 
	NOTE: In the process of grinding, using an air pump prevents the grinding solution from siphoning.</li>
	<li>Put the ground needles into a storage case. 
	NOTE: Use freshly ground needles as much as possible, though the ground needles can be kept in a storage case for at least a few weeks before microinjection.</li>
</ol>
4. Preparing pipettes for loading plasmids
<ol>
	<li>Heat a 200 &#181;L pipette with the outer flame of an alcohol burner.</li>
	<li>Once it starts to melt, stretch the pipette.</li>
	<li>Cut off the sealed end of the stretched pipette with scissors, and the stretched pipette is then ready to load DNA into a ground needle. 
	NOTE: Alternatively, extremely long and fine tips for filling ground needles are commercially available.</li>
	<li>Place these pipettes in an autoclaved plastic box.</li>
</ol>
5. Collecting embryos
<ol>
	<li>Collect flies (vas-phiC31; + ; attP2) expressing phiC31 integrase and bearing the attP2 docking site 3 days after they are hatched from 50 vials and transfer them to a cage (&#966; 90 mm x 150 mm). Add a little fresh yeast paste to the center of each grape agar plate18.</li>
	<li>Prepare 2-4 cages of flies. Keep the cages and plates at 25 &#176;C and change the plates every day for 2-4 days. 
	NOTE: This study combined the vas-phiC31 transgene (Stock # 36313, Bloomington Drosophila Stock Center) on the first chromosome and the attP2 transgene (Stock # 36313, Bloomington Drosophila Stock Center) on the third chromosome. The resulting stock (vas-phiC31; + ; attP2) is used in this protocol. Yeast paste needs to be prepared daily, and fresh yeast paste must be fed to the flies. The replaced cages need to be cleared and dried to get rid of the remaining embryos in the cages.</li>
	<li>On the day of microinjection, change the cages and plates in the morning. Then, change the plates every 30 min and collect embryos from these plates. Change all the cages and plates at room temperature (RT) and afterward keep the cages and plates at 25 &#176;C. 
	NOTE: Some protocols prefer to incubate the cages and plates at 18 &#176;C for egg laying. It depends on the number of embryos one needs for microinjection.</li>
</ol>
6. Dechorionating embryos
<ol>
	<li>Prepare 30% bleach by mixing 60 mL of bleach (containing active chlorine content of 34-46 g/L) and 140 mL of double distilled water.</li>
	<li>Remove the remaining flies on the grape juice agar plates with a brush or forceps, and rinse embryos into a cell strainer with double distilled water.</li>
	<li>Dry the cell strainer containing the embryos with tissue paper.</li>
	<li>Place it in a Petri dish containing 30% bleach, and swirl the cell strainer by hand or a horizontal shaker for 1.5 min at 60 rpm. 
	NOTE: Rinsing is a critical step. If the rinsing time is not long enough, the shell of an embryo will not come off completely. If the rinsing time is too long, it will cause the embryo to become soft and break easily. The cell strainer should be completely submerged in the 30% bleach. Wear a pair of rubber gloves and a lab coat to avoid injuries caused by bleach.</li>
	<li>Rinse embryos for 2 min with double distilled water stored in a laboratory plastic wash bottle to remove the residual bleach. Pick the floating embryos for the line-up.</li>
</ol>
7. Lining up embryos
<ol>
	<li>Cut the solid grape juice agar into long strips using a blade. 
	NOTE: Use purple grape juice to make it easier to line up white embryos.</li>
	<li>Transfer the embryos onto a long strip using a paintbrush.</li>
	<li>Line up around 60 embryos with a dissecting needle along the edge of a long strip with their posteriors facing the outside of the strip. Line up around 60 embryos within 5 min. 
	NOTE: The micropyle is a cone-shaped protrusion at the anterior pole of the embryo, through which the sperm enters to fertilize the oocyte (Figure 2A). Therefore, based on the cone-shaped protrusion at the anterior pole of the embryo, it is easy to determine the posterior of the dechorionated embryo.</li>
	<li>Place an 18 mm double-sided tape lengthwise along the edge of a coverslip (18 mm x 18 mm) and remove the paper backing. Gently press the coverslip onto the aligned embryos. Ensure the embryos are glued to the center of the tape.</li>
</ol>
8. Desiccating embryos
<ol>
	<li>Place the coverslip with the lined-up embryos in a box containing desiccant. The drying time varies depending on temperature and humidity. 
	NOTE: The drying time is critical for microinjection and needs to be experimentally determined. The drying time is usually 9-10 min in summer and 17-18 min in winter.</li>
	<li>Cover the embryos with halocarbon oil. 
	NOTE: Do not drop too much oil onto the embryos, as the extra oil may overflow the adhesive. The volume ratio of halocarbon oil 700 and halocarbon oil 27 is 1:3.</li>
</ol>
9. Microinjecting embryos
<ol>
	<li>Load 2 &#181;L of DNA into the needle by using the stretched pipette. Ensure that there are no bubbles in the needle. Place the needle into the needle holder of the micromanipulator.</li>
	<li>Place the aligned embryos covered with halocarbon oil under an inverted microscope. Ensure that the posterior ends of the embryos face the needle.</li>
	<li>Use the micromanipulator to bring the needle close to the embryos. Clear the needle. Ensure that a drop of liquid coming out of the needle is visible. Starting from 300 hPa injection pressure and 20 hPa equilibrium pressure, adjust the pressure of injection and the pressure of equilibrium to ensure the optimal size of the drop of liquid.</li>
	<li>Use the micromanipulator to insert the needle into the posterior ends of the embryos, and use the foot control connected to the microinjector to inject the DNA (Figure 2). If a small halo is visible, DNA is successfully injected into the embryo. 
	NOTE: Select the multinucleate embryos for injection and kill the older embryos using the &#34;clean&#34; button. If a large amount of cytoplasm leaks after injection, embryos are under-dried. If only a small amount of or no amount of cytoplasm leaks after injection, injections are considered successful. If more than one-third of the embryos leak cytoplasm after injection, extend the drying time by 1-2 min. If more than one-third of the embryos are over-dried, shorten the drying time by 1-2 min.</li>
	<li>Remove the needle from the embryo. Move the needle to the posterior end of the next embryo. Repeat the above steps.</li>
	<li>Put the injected embryos into an agar plate. Place the plate in a room at 18-20 &#176;C. On the next day, transfer the plate to an incubator at a temperature of 25 &#176;C and a humidity of 50%-60%, and incubate for 1 day. 
	NOTE: After injection, keep embryos at a temperature of 18-20 &#176;C to slow down their development, facilitate the integration of plasmid DNA into the Drosophila genome, and increase the survival rate of the injected embryos.</li>
</ol>
10. Collecting larvae
<ol>
	<li>Use a dissecting needle to scrape and loosen the food in a vial. Supply the food with a few drops of distilled water. 
	NOTE: Allow the distilled water to be absorbed into the food to prevent larvae from drowning.</li>
	<li>Take out the plates from the incubator.</li>
	<li>Collect the hatched larvae with a paintbrush under a stereo microscope and transfer them to the vial.</li>
	<li>Place the vial into an incubator at a temperature of 25 &#176;C and a humidity of 50%-60%.</li>
</ol>
11. Obtaining transgenic flies
<ol>
	<li>Collect the hatched flies after the vial is incubated at 25 &#176;C for 10 days.</li>
	<li>Cross a single male fly to three virgin female flies of the TM2/TM6C Sb1 Tb1 balancers, and cross a single female fly to three male flies of the TM2/TM6C Sb1 Tb1 balancers.</li>
	<li>Place the crosses into an incubator at a temperature of 25 &#176;C and a humidity of 50%-60%.</li>
	<li>Screen the progeny for flies with orange eyes. These are the transgenic flies (Figure 3A-C). 
	NOTE: Flies bearing a copy of transgene at the attP2 docking site have orange eyes.</li>
	<li>Cross a single orange-eyed male fly to three virgin female flies of the TM2/TM6C Sb1 Tb1 balancers to establish a stock. 
	NOTE: More than one generation of crosses need to be set up in order to cross out the phiC31 transgene on the first chromosome.</li>
</ol>

phiC31 Integrase-Mediated Embryo Transgenesis in Drosophila

<ol>
	<li>Rubin, G. M., Spradling, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+transformation+of+Drosophila+with+transposable+element+vectors.">Genetic transformation of Drosophila with transposable element vectors.</a> Science. 218 (4570), 348-353 (1982).</li><li>Venken, K. J., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenesis+upgrades+for+Drosophila+melanogaster.">Transgenesis upgrades for Drosophila melanogaster.</a> Development. 134 (20), 3571-3584 (2007).</li><li>Groth, A. C., Fish, M., Nusse, R., Calos, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+transgenic+Drosophila+by+using+the+site-specific+integrase+from+phage+phic31.">Construction of transgenic Drosophila by using the site-specific integrase from phage phic31.</a> Genetics. 166 (4), 1775-1782 (2004).</li><li>Bischof, J., Maeda, R. K., Hediger, M., Karch, F., Basler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+transgenesis+system+for+Drosophila+using+germ-line-specific+phic31+integrases.">An optimized transgenesis system for Drosophila using germ-line-specific phic31 integrases.</a> Proc Natl Acad Sci U S A. 104 (9), 3312-3317 (2007).</li><li>Markstein, M., Pitsouli, C., Villalta, C., Celniker, S. E., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploiting+position+effects+and+the+gypsy+retrovirus+insulator+to+engineer+precisely+expressed+transgenes.">Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes.</a> Nat Genet. 40 (4), 476-483 (2008).</li><li>Bateman, J. R., Lee, A. M., Wu, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+transformation+of+Drosophila+via+phic31+integrase-mediated+cassette+exchange.">Site-specific transformation of Drosophila via phic31 integrase-mediated cassette exchange.</a> Genetics. 173 (2), 769-777 (2006).</li><li>Venken, K. J., He, Y., Hoskins, R. A., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P[acman]:+A+BAC+transgenic+platform+for+targeted+insertion+of+large+DNA+fragments+in+D.+melanogaster.">P[acman]: A BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster.</a> Science. 314 (5806), 1747-1751 (2006).</li><li>Szabad, J., Bellen, H. J., Venken, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+assay+to+detect+in+vivo+Y+chromosome+loss+in+Drosophila+wing+disc+cells.">An assay to detect in vivo Y chromosome loss in Drosophila wing disc cells.</a> G3. 2 (9), 1095-1102 (2012).</li><li>Ringrose, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenesis+in+Drosophila+melanogaster.">Transgenesis in Drosophila melanogaster.</a> Methods Mol Biol. 561, 3-19 (2009).</li><li>Van Der Graaf, K., Srivastav, S., Singh, P., Mcnew, J. A., Stern, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+melanogaster+attp40+docking+site+and+derivatives+are+insertion+mutations+of+msp-300.">The Drosophila melanogaster attp40 docking site and derivatives are insertion mutations of msp-300.</a> PLoS One. 17 (12), e0278598 (2022).</li><li>Duan, Q., Estrella, R., Carson, A., Chen, Y., Volkan, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+Drosophila+attp40+background+on+the+glomerular+organization+of+or47b+olfactory+receptor+neurons.">The effect of Drosophila attp40 background on the glomerular organization of or47b olfactory receptor neurons.</a> G3. 13 (4), 022 (2023).</li><li>Dietzl, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+transgenic+RNAi+library+for+conditional+gene+inactivation+in+Drosophila.">A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila.</a> Nature. 448 (7150), 151-156 (2007).</li><li>Pfeiffer, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+for+neuroanatomy+and+neurogenetics+in+Drosophila.">Tools for neuroanatomy and neurogenetics in Drosophila.</a> Proc Natl Acad Sci U S A. 105 (28), 9715-9720 (2008).</li><li>Bellen, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+gene+disruption+project:+Progress+using+transposons+with+distinctive+site+specificities.">The Drosophila gene disruption project: Progress using transposons with distinctive site specificities.</a> Genetics. 188 (3), 731-743 (2011).</li><li>Zirin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+transgenic+drosophila+resource+collections+for+loss-+and+gain-of-function+studies.">Large-scale transgenic drosophila resource collections for loss- and gain-of-function studies.</a> Genetics. 214 (4), 755-767 (2020).</li><li>Wei, P., Xue, W., Zhao, Y., Ning, G., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-based+modular+assembly+of+a+UAS-cDNA/ORF+plasmid+library+for+more+than+5500+Drosophila+genes+conserved+in+humans.">CRISPR-based modular assembly of a UAS-cDNA/ORF plasmid library for more than 5500 Drosophila genes conserved in humans.</a> Genome Res. 30 (1), 95-106 (2020).</li><li>Fish, M. P., Groth, A. C., Calos, M. P., Nusse, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+transgenic+Drosophila+by+microinjecting+the+site-specific+phic31+integrase+mRNA+and+a+transgene-containing+donor+plasmid.">Creating transgenic Drosophila by microinjecting the site-specific phic31 integrase mRNA and a transgene-containing donor plasmid.</a> Nat Protoc. 2 (10), 2325-2331 (2007).</li><li>Fujioka, M., Jaynes, J. B., Bejsovec, A., Weir, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+transgenic+Drosophila.">Production of transgenic Drosophila.</a> Methods Mol Biol. 136, 353-363 (2000).</li><li>Loncar, D., Singer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+membrane+formation+during+the+cellularization+of+the+syncytial+blastoderm+of+Drosophila.">Cell membrane formation during the cellularization of the syncytial blastoderm of Drosophila.</a> Proc Natl Acad Sci U S A. 92 (6), 2199-2203 (1995).</li><li>Carmo, C., Araujo, M., Oliveira, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+techniques+in+fly+embryos+to+study+the+function+and+dynamics+of+SMC+complexes.">Microinjection techniques in fly embryos to study the function and dynamics of SMC complexes.</a> Methods Mol Biol. 2004, 251-268 (2019).</li></ol>

The authors have nothing to disclose.

Here, we present a protocol for embryo microinjection for transgenesis in Drosophila. For phiC31-mediated site-specific transgenesis, we injected 120-140 embryos for each plasmid and obtained at least one transgenic line for around 85% of plasmids (Supplementary Table 2). In our experience, plasmid DNA isolated by a plasmid miniprep kit suffices for transgenesis in Drosophila. Plasmid DNA concentrations ranging from 20 ng/&#181;L to 1683 ng/&#181;L might have no apparent effect on the success rate of transgenesis because most of them can produce transgenic flies (Supplementary Table 1).
The preparation of injection needles is critical for efficient embryo microinjection. An Injection needle is closed after it is pulled by a needle puller. For routine phiC31-mediated transgenesis in Drosophila, the tip of a needle is usually broken by a coverslip17. However, the randomly broken needles are often not well-suited for embryo microinjection, as they may get blocked or cause leakage of a large amount of cytoplasm and thereby have to be replaced by a new one during injection. Injection needles whose tips are beveled by a grinder do not usually get blocked or elicit large cytoplasmic leakage, which reduces the frequency of needle replacement. We noticed that the grinding solution can enter the beveled tip of a needle once the tip is open during grinding. Once it enters the needle, the grinding solution will not quickly dry up naturally. To prevent the grinding solution from siphoning, we connect an injection needle to a foot air pump with a pressure gauge. While grinding the tip of the needle, we use the air pump to apply air pressure to the needle, and thereby, the grinding solution is prevented from entering the beveled tip, enabling the beveled needle to be used immediately. Together, the foot air pump with the pressure gauge should help to accelerate transgenesis in Drosophila, particularly for a large of number of plasmids.
Identifying embryos at the pre-blastoderm or syncytial blastoderm stage is critical for microinjection19,20, as injecting embryos at later stages barely produces transgenic flies. Flies less than 10 days old are used for egg laying as they tend to lay more eggs than older ones. In the process of dechorionating embryos, a cell strainer is utilized to simplify the bleach treatment and double distilled water rinsing of embryos. After embryo rinsing, the floating embryos indicate that the eggshells have been well treated and can be used for subsequent microinjection. Both the dechorionation and drying time of embryos are critical for microinjection and should be optimized experimentally, as prolonged dechorionation or drying time can kill embryos. The dechorionation and drying time are affected by the temperature and humidity that vary from place to place and from time to time and should be adjusted accordingly based on the embryonic hatching rate.

The fruit fly Drosophila melanogaster is extremely amenable to genetic manipulation and genetic analysis. Transgenic fruit flies are widely used in biological research. Since it was developed in the early 1980s, P-element transposon-mediated transgenesis has been indispensable for Drosophila research1. In some scenarios, other transposons, such as piggyBac and Minos have been used for transgenesis in Drosophila as well2. Transgenes via transposon are randomly inserted into the Drosophila genome, and the expression levels of transgenes at different genomic loci may vary due to position effects and thereby can not be compared2. These drawbacks were overcome by the phiC31-mediated site-specific transgenesis3. The bacteriophage&#160;phiC31 gene encodes an integrase that mediates sequence-specific recombination between the attB and attP sites in Drosophila3. Many attP docking sites have been characterized and are available in the Bloomington Drosophila Stock Center3,4,5,6,7,8,9.
The phiC31 transgenesis system for Drosophila has been widely used by the fly community. Among the attP docking sites, attP18 on the X chromosome, attP40 on the second chromosome, and attP2 on the third chromosome are usually the preferred sites for transgene integration on each chromosome because transgenes at the three sites show high levels of inducible expression while having low levels of basal expression5. Recently, however, van der Graaf and colleagues found that the attP40 site, close to the Msp300 gene locus, and transgenes integrated at attP40 cause larval muscle nuclear clustering in Drosophila10. In another recent study, Duan and colleagues found that the homozygous attP40 chromosome disrupts the normal glomerular organization of the Or47b olfactory receptor neuron class in Drosophila11. These findings suggest that rigorous controls should be included when designing experiments and interpreting data.
Drosophila is an ideal model organism for in vivo interrogation of gene function due to its short life cycle, low cost, and easy maintenance. Genome-wide genetic screening relies on the construction of genome-wide transgenic libraries in Drosophila12,13,14,15. We previously generated 5551 UAS-cDNA/ORF constructs based on the binary GAL4/upstream activating sequence (GAL4/UAS) system, covering 83% of the Drosophila genes conserved in humans in the Drosophila Genomics Resource Center (DGRC) Gold Collection16. The UAS-cDNA/ORF plasmids can be used for the creation of a transgenic UAS-cDNA/ORF library in Drosophila. Efficient embryo microinjection technology can accelerate the creation of transgenic fly libraries.
For embryo microinjection, the needle tip is normally broken against a coverslip17. Thereby, needles frequently become clogged or cause leakage of large amounts of cytoplasm, and when these issues arise, new needles must be employed. Although beveling needles can enhance microinjection efficiency, the grinding solution enters the beveled tip during the grinding process. The grinding solution in the beveled tip does not quickly evaporate naturally; therefore, needles can not be utilized for microinjection directly after beveling. These factors reduce the efficiency of embryo microinjection procedures. Here, using the phiC31-mediated site-specific transgenesis in Drosophila as an example, we present a protocol for embryo microinjection for transgenesis in Drosophila.&#160;To prevent the grinding solution from entering the needle, we utilize an air pump to exert air pressure within the needle, thereby preventing the grinding solution from entering the beveled tip. Beveled needles are ready for sample loading and microinjection directly after beveling.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 &#956;m Cell Strainer</td>
			<td>NEST</td>
			<td>258367</td>
			<td></td>
		</tr>
		<tr>
			<td>3M double-sided adhesive</td>
			<td>3M</td>
			<td>415</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass</td>
			<td>SUTTER</td>
			<td>B100-75-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond abrasive plate</td>
			<td>SUTTER</td>
			<td>104E</td>
			<td></td>
		</tr>
		<tr>
			<td>FLAMING/BROWN Micropipette Puller</td>
			<td>SUTTER</td>
			<td>P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Foot air pump with pressure gauge</td>
			<td>Shenfeng</td>
			<td>SF8705D</td>
			<td></td>
		</tr>
		<tr>
			<td>Halocarbon oil 27</td>
			<td>Sigma</td>
			<td>H8773</td>
			<td></td>
		</tr>
		<tr>
			<td>Halocarbon oil 700</td>
			<td>Sigma</td>
			<td>H8898</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>ECLIPSE Ts2R</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjection pump</td>
			<td>eppendorf</td>
			<td>FemtoJet 4i</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>eppendorf</td>
			<td>TransferMan 4r</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette Beveler</td>
			<td>SUTTER</td>
			<td>BV-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover glasses (18 mm x 18 mm)</td>
			<td>CITOLAS</td>
			<td>10211818C</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides (25 mm x 75 mm)</td>
			<td>CITOLAS</td>
			<td>188105W</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish (90 mm x 15 mm)</td>
			<td>LAIBOER</td>
			<td>4190152</td>
			<td></td>
		</tr>
		<tr>
			<td>Photo-Flo 200</td>
			<td>Kodak</td>
			<td>1026269</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Nikon</td>
			<td>SMZ745</td>
			<td></td>
		</tr>
	</tbody>
</table>

embryo microinjection for transgenesis in drosophila

Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the construction of transgenic flies. Microinjection requires some types of equipment, including a microinjector, a micromanipulator, an inverted microscope, and a stereo microscope.&#160;Plasmids isolated with a plasmid miniprep kit are qualified for microinjection. Embryos at the pre-blastoderm or syncytial blastoderm stage, where nuclei share a common cytoplasm, are subjected to microinjection. A cell strainer eases the process of dechorionating embryos. The optimal time for dechorionation and desiccation of embryos needs to be determined experimentally. To increase the efficiency of embryo microinjection, needles prepared by a puller need to be beveled by a needle grinder. In the process of grinding needles, we utilize a foot air pump with a pressure gauge to avoid the capillary effect of the needle tip. We routinely inject 120-140 embryos for each plasmid and obtain at least one transgenic line for around 85% of plasmids. This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and&#160;presents a detailed protocol for embryo microinjection for transgenesis in Drosophila.

The tip of an injection needle is beveled by a needle grinder (Figure 1). One can bevel 50-60 needles in one hour. DNA is microinjected into the posterior of an embryo at the pre-blastoderm or syncytial blastoderm stage, where nuclei share a common cytoplasm (Figure 2A). The posterior of an embryo can be readily located based on the micropyle at the anterior of the embryo (Figure 2A). Embryos at the cellular blastoderm and gastrulation stages can not be used for injection, as each nucleus is encircled by a cell membrane at these stages, and thereby, the chance of obtaining transgenic flies is almost zero (Figure 2B). Overdried embryos are deformed when punctured and can not be used for microinjection (Figure 2C). Underdried embryos can extrude a large amount of cytoplasm when poked with the needle (Figure 2D), resulting in a lower survival rate of embryos. Embryos that are appropriately dried may or may not leak out a small amount of cytoplasm when injected (Figure 2E).
The Drosophila line expressing phiC31 integrase and bearing the attP2 docking site that is used for embryo microinjection in this protocol has white eyes (Figure 3A), and the aged flies of this genotype have pink eyes resulting from accumulated red fluorescent protein expressed specifically in the eye (Figure 3B). We use the mini-white gene as a selective marker for transgenesis in Drosophila, and transgenic flies are identified by eye color ranging from light yellow to red. Transgenic flies bearing a transgene at the attP2 docking site have orange eyes (Figure 3C), which one can tell readily from flies bearing no transgenes.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66679/66679fig01.jpg" /> 
Figure 1: Grinding the needles. (A) A foot air pump with a pressure gauge is utilized to apply air pressure to the needle. (B) A needle is mounted on a needle grinder at an angle of 35&#176; to a grinding plate surface. (C) Use a coarse adjustment knob to position the needle tip just above the grinding plate surface, then use a fine adjustment knob to start beveling the tip. (D) The tip of a pulled needle is under a microscope. Scale bars = 10 &#181;m.&#160;(E) A beveled needle is under a microscope. The beveled tip of the needle is indicated by an arrow. Scale bars = 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66679/66679fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66679/66679fig02.jpg" /> 
Figure 2: Drosophila embryos suitable for microinjection. (A) A Drosophila embryo at the pre-blastoderm or syncytial blastoderm stage can be injected in the posterior pole of the embryo, denoted by a red ellipse. The micropyle at the anterior pole of the embryo is indicated by an arrow. (B) A Drosophila embryo at the cellular blastoderm stage can not be used for microinjection. (C) An embryo that is overdesiccated looks deformed. (D) Insufficient desiccation of an embryo leads to a large amount of cytoplasmic leakage after injection. (E) For a successful injection, a small amount of cytoplasm may leak. Scale bars = 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66679/66679fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66679/66679fig03.jpg" /> 
Figure 3: Eye color of transgenic Drosophila bearing a transgene at the attP2 docking site. (A) The attP2 injection line expressing phiC31 integrase (vas-phiC31; + ; attP2) has a white eye. RFP specifically expressed in the eye is used as a selective marker for phiC31 transgene. (B) The aged attP2 injection line expressing phiC31 integrase has a pink eye resulting from the accumulated RFP protein in the eye. (C) Flies bearing a transgene integrated into the attP2 docking site have an orange eye. Scale bars = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66679/66679fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Table 1: Transgenesis in Drosophila resulting from embryo microinjection of 100 distinct plasmids. A total of 100 distinct plasmids of various concentrations ranging from 20 ng/&#181;L to 1683 ng/&#181;L were injected into embryos, and transgenic flies were obtained for 85 of the 100 plasmids. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66679/Supplementary Table 1.xlsx" target="_blank">Please click here to download this File.</a>
Supplementary Table 2: Efficiency of embryo microinjection for transgenesis in Drosophila. A total of 120 distinct plasmids were injected into embryos, and transgenic flies were obtained for 101 of the 120 plasmids. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66679/Supplementary Table 2.xlsx" target="_blank">Please click here to download this File.</a>

Author Spotlight: Optimizing Embryo Microinjection for Transgenesis in Drosophila

This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and presents an optimized protocol for embryo microinjection,&#160;a crucial step for creating transgenic flies.

Embryo Microinjection for Transgenesis in Drosophila

Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the ...

embryo-microinjection-for-transgenesis-in-drosophila

Nanyang Technological University

Research

JoVE Journal

Genetics

1.6K Views.  University of South China. 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.

Video: Author Spotlight: Optimizing Embryo Microinjection for Transgenesis in Drosophila

Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can create genetic modifications that will be present in every cell of an adult organism. It can also be used to deliver siRNA, mRNAs or blocking antibodies to study gene function in preimplantation embryos. The conventional technique for microinjecting embryos used in rodents consists of a very thin micropipette that directly penetrates the plasma membrane when advanced into the embryo. When this technique is applied to livestock animals it usually results in low efficiency. This is mainly because in contrast to mice and rats, bovine, ovine, and porcine zygotes have a very dark cytoplasm and a highly elastic plasma membrane that makes visualization during injection and penetration of the plasma membrane hard to achieve. In this protocol, we describe a suitable microinjection method for the delivery of solutions into the cytoplasm of cattle zygotes that has proved to be successful for sheep and pig embryos as well. First, a laser is used to create a hole in the zona pellucida. Then a blunt-end glass micropipette is introduced through the hole and advanced until the tip of the needle reaches about 3/4 into the embryo. Then, the plasma membrane is broken by aspiration of cytoplasmic content inside the needle. Finally, the aspirated cytoplasmic content followed by the solution of interest is injected back into the embryonic cytoplasm. This protocol has been successfully used for the delivery of different solutions into bovine and ovine zygotes with 100% efficiency, minimal lysis, and normal blastocysts development rates.

This protocol shows how to perform cytoplasmic microinjection in farm animal zygotes. This technique can be used to deliver any solution into the one-cell embryo such as genome editing tools to generate knockout animals.

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can ...

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. This is due to its reproductive behavior: females damage the external surface of fruits and vegetables when they lay eggs and the hatched larvae feed on their pulp. Wild C. capitata populations are traditionally controlled through insecticide spraying and/or eco-friendly approaches, the most successful being the Sterile Insect Technique (SIT). The SIT relies on mass-rearing, radiation-based sterilization and field release of males that retain their capacity to mate but are not able to generate fertile progeny. The advent and the subsequent rapid development of biotechnological tools, together with the availability of the medfly genome sequence, has greatly boosted our understanding of the biology of this species. This favored the proliferation of new strategies for genome manipulation, which can be applied to population control.
In this context, embryo microinjection plays a dual role in expanding the toolbox for medfly control. The ability to interfere with the function of genes that regulate key biological processes, indeed, expands our understanding of the molecular machinery underlying medfly invasiveness. Furthermore, the ability to achieve germ-line transformation facilitates the production of multiple transgenic strains that can be tested for future field applications in novel SIT settings. Indeed, genetic manipulation can be used to confer desirable traits that can, for example, be used to monitor sterile male performance in the field, or that can result in early life-stage lethality. Here we describe a method to microinject nucleic acids into medfly embryos to achieve these two main goals.

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae) is a worldwide pest of agriculture. A deeper understanding of its biology is key to control medfly populations and thus reduce economic impact. Embryo microinjection is a fundamental tool allowing both germ-line transformation and reverse genetics studies in this species.

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. ...

Quantifying Abdominal Pigmentation in Drosophila melanogaster

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for understanding the development and evolution of morphological phenotypes. Abdominal pigmentation in Drosophila melanogaster has been particularly useful, allowing researchers to identify the loci that underlie inter- and intraspecific variations in morphology. Hitherto, however, D. melanogaster abdominal pigmentation has been largely assayed qualitatively, through scoring, rather than quantitatively, which limits the forms of statistical analysis that can be applied to pigmentation data. This work describes a new methodology that allows for the quantification of various aspects of the abdominal pigmentation pattern of adult D. melanogaster. The protocol includes specimen mounting, image capture, data extraction, and analysis. All the software used for image capture and analysis feature macros written for open-source image analysis. The advantage of this approach is the ability to precisely measure pigmentation traits using a methodology that is highly reproducible across different imaging systems. While the technique has been used to measure variation in the tergal pigmentation patterns of adult D. melanogaster, the methodology is flexible and broadly applicable to pigmentation patterns in myriad different organisms.

Quantifying Abdominal Pigmentation in Drosophila melanogaster

This work presents a method to quickly and precisely quantify the abdominal pigmentation of Drosophila melanogaster using digital image analysis. This method streamlines the procedures between phenotype acquisition and data analysis and includes specimen mounting, image acquisition, pixel value extraction, and trait measurement.

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for ...

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene function. Gene knockout has been used to study these gene functions in vivo. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a powerful tool used to edit genomic DNA sequences to alter gene function. While the traditional approach has been to introduce CRISPR/Cas9 mRNA into the single cell embryos through microinjection, this can be a slow and inefficient process in catfish. Here, a detailed protocol for microinjection of channel catfish embryos with CRISPR/Cas9 protein is described. Briefly, eggs and sperm were collected and then artificial fertilization performed. Fertilized eggs were transferred to a Petri dish containing Holtfreter's solution. Injection volume was calibrated and then guide RNAs/Cas9 targeting the toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) gene and rhamnose binding lectin (RBL) gene were microinjected into the yolk of one-cell embryos. The gene knockout was successful as indels were confirmed by DNA sequencing. The predicted protein sequence alterations due to these mutations included frameshift and truncated protein due to premature stop codons.

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

A simple and efficient microinjection protocol for gene editing in channel catfish embryos using the CRISPR/Cas9 system is presented. In this protocol, guide RNAs and Cas9 protein were microinjected into the yolk of one-cell embryos. This protocol has been validated by knocking out two channel catfish immune-related genes.

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

Microinjection of Nasonia vitripennis embryos is an essential method for generating heritable genome modifications. Described here is a detailed procedure for microinjection and transplantation of Nasonia vitripennis embryos, which will greatly facilitate future genome manipulation in this organism.

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex ...

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

The incidence of complex metabolic diseases has increased as a result of a widespread transition towards lifestyles of increased caloric intake and lowered activity levels. These multifactorial diseases arise from a combination of genetic, environmental, and behavioral factors. One such complex disease is Metabolic Syndrome (MetS), which is a cluster of metabolic disorders, including hypertension, hyperglycemia, and abdominal obesity. Exercise and dietary intervention are the primary treatments recommended by doctors to mitigate obesity and its subsequent metabolic diseases. Exercise intervention, in particular aerobic interval training, stimulates favorable changes in the common risk factors for Type 2 Diabetes Mellitus (T2DM), Cardiovascular Disease (CVD), and other conditions. With the influx of evidence describing the therapeutic effect exercise has on metabolic health, establishing a system that models exercise in a controlled setting provides a valuable tool for assessing the effects of exercise in an experimental context. Drosophila melanogaster is a great tool for investigating the physiological and molecular changes that result from exercise intervention. The flies have short lifespans and similar mechanisms of metabolizing nutrients when compared to humans. To induce exercise in Drosophila, we developed a machine called the TreadWheel, which utilizes the fly&#39;s innate, negative geotaxis tendency to gently induce climbing. This enables researchers to perform experiments on large cohorts of genetically diverse flies to better understand the genotype-by-environment interactions underlying the effects of exercise on metabolic health.

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

The TreadWheel uses rotational motion to gently induce exercise in adult Drosophila melanogaster by exploiting flies&#39; innate, negative geotaxis. It allows for analysis of the interactions between exercise and factors, such as genotype, sex, and diet, and their effect on physiological and molecular assays to assess metabolic health.

The incidence of complex metabolic diseases has increased as a result of a widespread transition towards lifestyles of increased caloric intake and ...

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human genetics research. Although a number of in silico algorithms have been developed to predict the pathogenicity of variants identified in these datasets, functional studies are critical to determining how specific genomic variants affect protein function, especially for missense variants. In the Undiagnosed Diseases Network (UDN) and other rare disease research consortia, model organisms (MO) including Drosophila, C. elegans, zebrafish, and mice are actively used to assess the function of putative human disease-causing variants. This protocol describes a method for the functional assessment of rare human variants used in the Model Organisms Screening Center Drosophila Core of the UDN. The workflow begins with gathering human and MO information from multiple public databases, using the MARRVEL web resource to assess whether the variant is likely to contribute to a patient&#39;s condition as well as design effective experiments based on available knowledge and resources. Next, genetic tools (e.g., T2A-GAL4 and UAS-human cDNA lines) are generated to assess the functions of variants of interest in Drosophila. Upon development of these reagents, two-pronged functional assays based on rescue and overexpression experiments can be performed to assess variant function. In the rescue branch, the endogenous fly genes are &#34;humanized&#34;&#160;by replacing the orthologous Drosophila gene with reference or variant human transgenes. In the overexpression branch, the reference and variant human proteins are exogenously driven in a variety of tissues. In both cases, any scorable phenotype (e.g., lethality, eye morphology, electrophysiology) can be used as a read-out, irrespective of the disease of interest. Differences observed between reference and variant alleles suggest a variant-specific effect, and thus likely pathogenicity. This protocol allows rapid, in vivo assessments of putative human disease-causing variants of genes with known and unknown functions.

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

The goal of this protocol is to outline the design and performance of in vivo experiments in Drosophila melanogaster to assess the functional consequences of rare gene variants associated with human diseases.

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and mechanobiology, which all influence muscle development and myofibrillogenesis. Omics data, such as those generated by mass spectrometry or deep sequencing, can provide important mechanistic insights into these biological processes. For such approaches, it is beneficial to analyze tissue-specific samples to increase both selectivity and specificity of the omics fingerprints. Here we present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched muscle samples for omics applications. We first describe how to dissect flight muscles at early pupal stages (&#60;48 h after puparium formation [APF]), when the muscles are discernable by green fluorescent protein (GFP) labeling. We then describe how to dissect muscles from late pupae (&#62;48 h APF) or adults, when muscles are distinguishable under a dissecting microscope. The accompanying video protocol will make these technically demanding dissections more widely accessible to the muscle and Drosophila research communities. For RNA applications, we assay the quantity and quality of RNA that can be isolated at different time points and with different approaches. We further show that Bruno1 (Bru1) is necessary for a temporal shift in myosin heavy chain (Mhc) splicing, demonstrating that dissected muscles can be used for mRNA-Seq, mass spectrometry, and reverse transcription polymerase chain reaction (RT-PCR) applications. This dissection protocol will help promote tissue-specific omics analyses and can be generally applied to study multiple biological aspects of myogenesis.

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study transcriptional regulation, alternative splicing, metabolism, and mechanobiology. We present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched samples ideal for proteomics and deep-sequencing. These samples can offer important mechanistic insights into diverse aspects of muscle development.

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and ...

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus

Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, Eastern equine encephalitis, lymphatic filariasis, and Saint Louis encephalitis. Notably, avian malaria has played a major role in the extinction of numerous endemic island bird species, while WNV has become an important vector-borne disease in the United States. To gain further insight into C. quinquefasciatus biology and expand their genetic control toolbox, we need to develop more efficient and affordable methods for genome engineering in this species. However, some biological traits unique to Culex mosquitoes, particularly their egg rafts, have made it difficult to perform microinjection procedures required for genome engineering. To address these challenges, we have developed an optimized embryo microinjection protocol that focuses on mitigating the technical obstacles associated with the unique characteristics of Culex mosquitoes. These procedures demonstrate optimized methods for egg collection, egg raft separation and other handling procedures essential for successful microinjection in C. quinquefasciatus. When coupled with the CRISPR/Cas9 genome editing technology, these procedures allow us to achieve site-specific, efficient and heritable germline mutations, which are required to perform advanced genome engineering and develop genetic control technologies in this important, but currently understudied, disease vector.

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus

This protocol describes microinjection procedures for Culex quinquefasciatus embryos that are optimized to work with CRISPR/Cas9 gene editing tools. This technique can efficiently generate site-specific, heritable, germline mutations that can be used for building genetic technologies in this understudied disease vector.

Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, ...