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.</...

phiC31 Integrase-Mediated Embryo Transgenesis in Drosophila

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	<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>

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 s...

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.

Author Spotlight: Optimizing Embryo Microinjection for Transgenesis in Drosophila

Embryo Microinjection for Transgenesis in Drosophila

Transgenesis in Drosophila is essential for studying gene function at the organism level. Embryo microinjection is a crucial step for construction of transgenic flies. Here, we take the phiC31 integrase-mediated transgenesis in Drosophila as an example and present a detailed protocol for embryo microinjection for transgenesis in Drosophila.Injection needles whose tips are beveled by a grinder do not usually get blocked or elicit large cytoplasmic leakage. However, the grinding solution can enter the beveled tip of a needle during the grinding process and the grinding solution inside the tip does not naturally dry up quickly. Our protocol uses a foot air pump with a pressure gauge to apply air pressure to a needle while grinding its tip.This prevents the grinding solution from siphoning, thereby the grinding solution is prevented from entering the beveled tip, enabling the beveled needle to be used immediately.

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 gastrulat...

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.

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