pPV540 and pPV402 were kind gifts from Dr. James Lok at the University of Pennsylvania. We thank Astra Bryant for helpful comments on the manuscript. This work was funded by a Burroughs-Wellcome Fund Investigators in the Pathogenesis of Disease Award, a Howard Hughes Medical Institute Faculty Scholar Award, and National Institutes of Health R01 DC017959 (E.A.H.).

<ol>
	<li>Krolewiecki, A. 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=A+public+health+response+against+Strongyloides+stercoralis:+time+to+look+at+soil-transmitted+helminthiasis+in+full.">A public health response against Strongyloides stercoralis: time to look at soil-transmitted helminthiasis in full.</a> PLoS Neglected Tropical Diseases. 7 (5), 2165 (2013).</li><li>Buonfrate, 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=The+global+prevalence+of+Strongyloides+stercoralis+infection.">The global prevalence of Strongyloides stercoralis infection.</a> Pathogens. 9 (6), 468 (2020).</li><li>Castelletto, M. L., Gang, S. S., Hallem, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+functional+genomics+for+parasitic+nematodes+of+mammals.">Recent advances in functional genomics for parasitic nematodes of mammals.</a> Journal of Experimental Biology. 223, 206482 (2020).</li><li>Evans, T. C., 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=Transformation+and+microinjection.">Transformation and microinjection.</a> WormBook. , (2006).</li><li>Lok, J. B., Unnasch, T. R., 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=Transgenesis+in+animal+parasitic+nematodes:+Strongyloides+spp.+and+Brugia+spp.">Transgenesis in animal parasitic nematodes: Strongyloides spp. and Brugia spp.</a> WormBook. , (2013).</li><li>Shao, H. G., Li, X. S., Lok, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heritable+genetic+transformation+of+Strongyloides+stercoralis+by+microinjection+of+plasmid+DNA+constructs+into+the+male+germline.">Heritable genetic transformation of Strongyloides stercoralis by microinjection of plasmid DNA constructs into the male germline.</a> International Journal for Parasitology. 47 (9), 511-515 (2017).</li><li>Schafer, T. W., Skopic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parasites+of+the+small+intestine.">Parasites of the small intestine.</a> Current Gastroenterology Reports. 8 (4), 312-320 (2006).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> The C. elegans Research Community, WormBook. , (2006).</li><li>Gang, S. S., 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=Targeted+mutagenesis+in+a+human-parasitic+nematode.">Targeted mutagenesis in a human-parasitic nematode.</a> PLoS Pathogens. 13 (10), 1006675 (2017).</li><li>Lok, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strongyloides+stercoralis:+a+model+for+translational+research+on+parasitic+nematode+biology.">Strongyloides stercoralis: a model for translational research on parasitic nematode biology.</a> The C. elegans Research Community, WormBook. , (2007).</li><li>Hawdon, J. M., Schad, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+storage+of+hookworm+infective+larvae+in+buffered+saline+solution+maintains+larval+responsiveness+to+host+signals.">Long-term storage of hookworm infective larvae in buffered saline solution maintains larval responsiveness to host signals.</a> Proceedings of the Helminthological Society of Washington (USA). 58 (1), 140-142 (1991).</li><li>Bargmann, C. I., Hartwieg, E., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant-selective+genes+and+neurons+mediate+olfaction+in+C.+elegans.">Odorant-selective genes and neurons mediate olfaction in C. elegans.</a> Cell. 74 (3), 515-527 (1993).</li><li>Junio, A. B., 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=Strongyloides+stercoralis:+cell-+and+tissue-specific+transgene+expression+and+co-transformation+with+vector+constructs+incorporating+a+common+multifunctional+3'+UTR.">Strongyloides stercoralis: cell- and tissue-specific transgene expression and co-transformation with vector constructs incorporating a common multifunctional 3' UTR.</a> Experimental Parasitology. 118 (2), 253-265 (2008).</li><li>Gang, S. S., 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=Chemosensory+mechanisms+of+host+seeking+and+infectivity+in+skin-penetrating+nematodes.">Chemosensory mechanisms of host seeking and infectivity in skin-penetrating nematodes.</a> Proceedings of the National Academy of Sciences of the United States of America. 117 (30), 17913-17923 (2020).</li><li>Bryant, A. S., 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+critical+role+for+thermosensation+in+host+seeking+by+skin-penetrating+nematodes.">A critical role for thermosensation in host seeking by skin-penetrating nematodes.</a> Current Biology. 28 (14), 2338-2347 (2018).</li><li>Lok, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleic+acid+transfection+and+transgenesis+in+parasitic+nematodes.">Nucleic acid transfection and transgenesis in parasitic nematodes.</a> Parasitology. 139 (5), 574-588 (2012).</li><li>Shao, H., 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=Transposon-mediated+chromosomal+integration+of+transgenes+in+the+parasitic+nematode+Strongyloides+ratti+and+establishment+of+stable+transgenic+lines.">Transposon-mediated chromosomal integration of transgenes in the parasitic nematode Strongyloides ratti and establishment of stable transgenic lines.</a> PLoS Pathogens. 8 (8), 1002871 (2012).</li><li>Lok, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=piggyBac:+a+vehicle+for+integrative+DNA+transformation+of+parasitic+nematodes.">piggyBac: a vehicle for integrative DNA transformation of parasitic nematodes.</a> Mobile Genetic Elements. 3 (2), 24417 (2013).</li><li>Li, X., 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=Successful+transgenesis+of+the+parasitic+nematode+Strongyloides+stercoralis+requires+endogenous+non-coding+control+elements.">Successful transgenesis of the parasitic nematode Strongyloides stercoralis requires endogenous non-coding control elements.</a> International Journal for Parasitology. 36 (6), 671-679 (2006).</li><li>Bryant, A. S., Hallem, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Wild+Worm+Codon+Adapter:+a+web+tool+for+automated+codon+adaptation+of+transgenes+for+expression+in+non-Caenorhabditis+nematodes.">The Wild Worm Codon Adapter: a web tool for automated codon adaptation of transgenes for expression in non-Caenorhabditis nematodes.</a> G3. 3 (7), (2021).</li><li>Crane, M., 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=In+vivo+measurements+reveal+a+single+5'-intron+is+sufficient+to+increase+protein+expression+level+in+Caenorhabditis+elegans.">In vivo measurements reveal a single 5'-intron is sufficient to increase protein expression level in Caenorhabditis elegans.</a> Scientific Reports. 9 (1), 9192 (2019).</li><li>Han, Z., 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=Improving+transgenesis+efficiency+and+CRISPR-associated+tools+through+codon+optimization+and+native+intron+addition+in+Pristionchus+nematodes.">Improving transgenesis efficiency and CRISPR-associated tools through codon optimization and native intron addition in Pristionchus nematodes.</a> Genetics. 216 (4), 947-956 (2020).</li><li>Adams, S., Pathak, P., Shao, H., Lok, J. B., Pires-daSilva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposome-based+transfection+enhances+RNAi+and+CRISPR-mediated+mutagenesis+in+non-model+nematode+systems.">Liposome-based transfection enhances RNAi and CRISPR-mediated mutagenesis in non-model nematode systems.</a> Scientific Reports. 9 (1), 483 (2019).</li><li>Dulovic, A., Puller, V., Streit, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+culture+conditions+for+free-living+stages+of+the+nematode+parasite+Strongyloides+ratti.">Optimizing culture conditions for free-living stages of the nematode parasite Strongyloides ratti.</a> Experimental Parasitology. 168, 25-30 (2016).</li><li>Harvey, S. C., Gemmill, A. W., Read, A. F., Viney, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+control+of+morph+development+in+the+parasitic+nematode+Strongyloides+ratti.">The control of morph development in the parasitic nematode Strongyloides ratti.</a> Proceedings of the Royal Society B: Biological Sciences. 267 (1457), 2057-2063 (2000).</li><li>Kim, A., Pyykko, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+matters:+versatile+use+of+PiggyBac+transposons+as+a+genetic+manipulation+tool.">Size matters: versatile use of PiggyBac transposons as a genetic manipulation tool.</a> Molecular and Cellular Biochemistry. 354 (1-2), 301-309 (2011).</li><li>Lok, J. B., Shao, H., Massey, H. C., Li, X. <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+Strongyloides+and+related+parasitic+nematodes:+historical+perspectives,+current+functional+genomic+applications+and+progress+towards+gene+disruption+and+editing.">Transgenesis in Strongyloides and related parasitic nematodes: historical perspectives, current functional genomic applications and progress towards gene disruption and editing.</a> Parasitology. 144 (3), 327-342 (2017).</li><li>Farboud, B., Meyer, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dramatic+enhancement+of+genome+editing+by+CRISPR/Cas9+through+improved+guide+RNA+design.">Dramatic enhancement of genome editing by CRISPR/Cas9 through improved guide RNA design.</a> Genetics. 199 (4), 959-971 (2015).</li><li>Cheong, M. C., 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=Identification+of+a+nuclear+receptor/coactivator+developmental+signaling+pathway+in+the+nematode+parasite+Strongyloides+stercoralis.">Identification of a nuclear receptor/coactivator developmental signaling pathway in the nematode parasite Strongyloides stercoralis.</a> Proceedings of the National Academy of Sciences of the United States of America. 118 (8), 2021864118 (2021).</li><li>Nolan, T. J., Megyeri, Z., Bhopale, V. M., Schad, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strongyloides+stercoralis:+the+first+rodent+model+for+uncomplicated+and+hyperinfective+strongyloidiasis,+the+Mongolian+gerbil+(Meriones+unguiculatus).">Strongyloides stercoralis: the first rodent model for uncomplicated and hyperinfective strongyloidiasis, the Mongolian gerbil (Meriones unguiculatus).</a> Journal of Infectious Diseases. 168 (6), 1479-1484 (1993).</li><li>Li, X., 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=Transgenesis+in+the+parasitic+nematode+Strongyloides+ratti.">Transgenesis in the parasitic nematode Strongyloides ratti.</a> Molecular and Biochemical Parasitology. 179 (2), 114-119 (2011).</li><li>Viney, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploiting+the+life+cycle+of+Strongyloides+ratti.">Exploiting the life cycle of Strongyloides ratti.</a> Parasitology Today. 15 (6), 231-235 (1999).</li><li>Stoltzfus, J. D., Massey, H. C., Nolan, T. J., Griffith, S. D., Lok, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strongyloides+stercoralis+age-1:+a+potential+regulator+of+infective+larval+development+in+a+parasitic+nematode.">Strongyloides stercoralis age-1: a potential regulator of infective larval development in a parasitic nematode.</a> PLoS ONE. 7 (6), 38587 (2012).</li><li>Castelletto, M. L., Massey, H. C., Lok, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+of+Strongyloides+stercoralis+infective+larvae+requires+the+DAF-16+ortholog+FKTF-1.">Morphogenesis of Strongyloides stercoralis infective larvae requires the DAF-16 ortholog FKTF-1.</a> PLoS Pathogens. 5 (4), 1000370 (2009).</li><li>Douglas, B., 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=Transgenic+expression+of+a+T+cell+epitope+in+Strongyloides+ratti+reveals+that+helminth-specific+CD4++T+cells+constitute+both+Th2+and+Treg+populations.">Transgenic expression of a T cell epitope in Strongyloides ratti reveals that helminth-specific CD4+ T cells constitute both Th2 and Treg populations.</a> PLoS Pathogens. 17 (7), 1009709 (2021).</li></ol>

The authors declare no conflicts of interest.

This microinjection protocol details the methods for introducing constructs for transgenesis and CRISPR/Cas9-mediated mutagenesis into S.&#160;stercoralis and S.&#160;ratti. For both S.&#160;stercoralis and S.&#160;ratti, post-injection survival and the rate of transgenesis or mutagenesis are subject to several variables that can be fine-tuned.
The first critical consideration for successful transgenesis is how plasmid transgenes are constructed. Previous studies have found that expression of exogenous transgenes in Strongyloides requires the use of Strongyloides 5&#39; promoters and 3&#39; UTR elements3,13,19. Similar to C. elegans constructs, Strongyloides constructs generally use a gene-specific promoter and a common 3&#39; UTR, such as the one from the Ss-era-1 gene13. Codon-optimization of the coding region may also be important for expression in Strongyloides. Recently, the Wild Worm Codon Adaptor, a web-based app that codon-optimizes coding sequences for Strongyloides and other nematodes, was developed20. Finally, while not rigorously tested in Strongyloides, introns have been shown to increase expression of exogenous transgenes in both C. elegans21 and the insect-associated nematode Pristionchus pacificus22&#160;and are presumed to increase expression in Strongyloides as well. The Wild Worm Codon Adaptor has options for including up to three introns in the modified sequence20.
The composition and delivery of the microinjection mix affect the transgenesis rate and the survival of the F1 progeny. BU saline is routinely used as the diluent for mixes, although using ddH2O is also an option. The concentrations and/or ratio of components in the mix can be adjusted to improve the transformation rate. Higher concentrations of the plasmids of interest can increase the rate of transgenesis but often result in fewer total progeny. If no transgene expression is observed or only dead transgenic eggs are found, it is possible that the transgenes are toxic, or that something in the plasmid stocks is causing the death of the transgenics. In the latter case, making new plasmid stocks using a different method (for example, using a different miniprep kit) may be sufficient for obtaining transgenics. Adding lipofectamine to the microinjection mix may also improve the rate of transgenesis23.
The shape of the microinjection needle delivering the mix also affects the survival and transgenesis rates (Figure 3A-F, Video 1, Video 2, and Supplemental Video S1). The needle must be sharp enough to penetrate the cuticle and narrow enough to not result in excessive damage. It is recommended to pull needles just before use as needles stored for more than a day may accumulate debris and become clogged during microinjections. Recovering injected females from the microinjection pad without damaging them can be accomplished with a few different methods. One technique is to float the worms off the injection pad in a drop of BU, and then use HB101 on a worm pick to collect the worms. Other techniques for recovery include floating the worms in BU and collecting them using a pipette tip or a small paintbrush or simply using a worm pick alone to move the worms to a recovery plate.
If no progeny were obtained from the microinjected females, this suggests that either the injected females were damaged in the microinjection process or the post-injection culturing conditions were suboptimal. There are a number of different post-injection culturing conditions that can be tried. The small-scale fecal-charcoal cultures described above generally support better worm survival than NGM plates with HB101. However, it can be difficult to follow the survival of the injected worms and the development of the F1 larvae on fecal-charcoal plates, and eggs are not visible on these plates. An advantage of culturing worms on NGM plates with HB101 instead of fecal-charcoal plates is to allow careful observation of egg-laying and larval development, which can be useful for troubleshooting. V12 plates with HB101 can also be used to increase survival24. Finally, a chemotaxis plate12 with a single rat fecal pellet can be used for S.&#160;ratti post-injection culturing. The males and injected females are transferred directly to the rat fecal pellet. In 5-7 days, worms are collected from the agar and feces using a Baermann apparatus, as described above.
To obtain Strongyloides adults for microinjection, freshly prepared fecal-charcoal plates can be incubated at 25 &#176;C for 24 h or 20 &#176;C for 48 h. Strongyloides adults reared at 25 &#176;C for 24 h are young enough to produce a large number of progeny25. However, if the females are too young, they may not survive the microinjection process. Adults collected from fecal-charcoal plates that have been incubated at 20 &#176;C for ~48 h are more likely to tolerate the microinjection process. However, because these adults are older than adults obtained from a 24 h incubation at 25&#160;&#176;C, they are not as fecund and may have a lower transformation rate. Novices may prefer to start with older adults and then switch to slightly younger adults as skills improve.
Like C. elegans, Strongyloides species can express transgenes from extrachromosomal arrays and genome-integrated constructs in the F1 generation. Unlike C. elegans, Strongyloides species will only express genome-integrated transgenes in the F2 and subsequent generations even though the extrachromosomal arrays are still detectable by PCR13. The F1 transgenic larvae expressing extrachromosomal arrays can be used for experiments that do not require genome integration or large numbers of transgenic worms. Genome integration may be required for experiments that require tagging endogenous genes or testing large numbers of worms in population-based assays. There are two methods for genome integration of transgenes in Strongyloides: piggyBac transposon-mediated integration17 and CRISPR/Cas9-mediated integration9. piggyBac transposon-mediated integration uses the piggyBac transposase to target cargo to TTAA sites in the genome26. Because the TTAA motif is quite common in the AT-rich genome of Strongyloides species, integration is often at more than one site in the genome. In contrast, CRISPR/Cas9-mediated integration can be used to integrate transgenes at a specific target locus9.
The CRISPR/Cas9 system can also be used to generate targeted gene knockouts9,27. Due to the AT richness of the Strongyloides genome, finding usable Cas9 target sites containing the optimal 5&#39;-N18GGNGG-3&#39; sequence for nematodes28 is a challenge. Frequently, there are only one or two sites in a gene for targeting. The preferred method involves the integration of a repair template containing a transgene with a fluorescent marker by homology-directed repair into the genomic locus, resulting in complete disruption of the gene. Potential knockouts can be identified by expression of the transgene9,14,15,29. However, transgene expression alone is not indicative of genotype as expression from arrays vs. integrated transgenes is often indistinguishable. Thus, the F1 transgenic larvae require post-hoc genotyping to identify homozygous knockouts9. In the absence of a repair template, mutagenesis of the target locus occurs at high frequency but can result in large deletions9.
Although generating transgenic or mutant F1 larvae is relatively straightforward in S.&#160;stercoralis, generating stable lines is extremely difficult because of the need for host passage. In the laboratory, the Mongolian gerbil is a permissive host for S.&#160;stercoralis but requires a high dose of worms to establish an infection capable of producing enough F2 larvae to establish the line30. Only approximately 6% of infective larvae become parasitic females30. Furthermore, if the transgenic larvae have array expression without genome integration, they will not produce the transgene-expressing progeny required to infect a second gerbil host. To increase the chances of sufficient numbers of genome-integrated larvae becoming reproductive parasitic adults, a minimum of 400-500 transgenic larvae in the initial inoculum is recommended. It may be possible to reduce the number of larvae required to establish a patent infection by treating the gerbils with prednisone30. Nevertheless, it is likely to be difficult to amass enough integrated transgenic or mutant worms to successfully establish a stable line of S.&#160;stercoralis. However, it is usually feasible to amass sufficient numbers of transgenic S.&#160;stercoralis F1 larvae for single-worm assays14,15.
Strongyloides ratti has the distinct advantage of the greater feasibility of generating stable transgenic or knockout lines17,31. S.&#160;ratti free-living adult females are less tolerant of the microinjection process than S.&#160;stercoralis free-living adult females; S.&#160;ratti females generally produce fewer overall larvae than S.&#160;stercoralis females, and the transformation rate is also lower31. However, only a few transgenic or knockout F1 larvae are required to establish a stable line of S.&#160;ratti. As S.&#160;ratti is a natural parasite of rats, only a few S.&#160;ratti infective larvae are sufficient to establish a patent infection32. Thus, it is generally possible to amass sufficient numbers of transgenic or mutant larvae to establish a stable line. Because Strongyloides species will not express extrachromosomal arrays past the F1 generation, only genome-integrated F1 larvae can produce a stable transgenic line13. It is generally impossible to identify worms with integrated transgenes prior to genotyping, so the protocol is to collect all transgenic F1 larvae and inject them into a rat. Some small percentage of these larvae will have the desired integration event; these larvae will form the basis for the stable line. As the piggyBac method often results in more than one integration event in any individual worm, almost 100% transmission of the transgene can be achieved after a few rounds of passaging transgenic larvae through a rat17.
In summary, the technique described here can be used to generate transgenic or knockout S.&#160;stercoralis and S.&#160;ratti. This enables a wide range of potential experiments, including but not limited to the cell-specific expression of transgenes, the generation of mutants, and the endogenous tagging of proteins to determine spatial and temporal functions14,15,29,33,34,35. In the long run, knowledge gained from the use of transgenic Strongyloides can be used to develop new strategies to combat human infections with S.&#160;stercoralis and other intestinal parasitic nematodes.

Strongyloides stercoralis has long been overlooked as an important human pathogen compared to the more widely recognized hookworms and the roundworm Ascaris lumbricoides1. Previous studies of worm burden often severely underestimated the prevalence of S.&#160;stercoralis due to the low sensitivity of common diagnostic methods for S.&#160;stercoralis2. In recent years, epidemiological studies based on improved diagnostic tools have estimated that the true prevalence of S.&#160;stercoralis infections is much higher than previously reported, approximately 610 million people worldwide2.
Both S.&#160;stercoralis and other Strongyloides species, including the closely related rat parasite and common lab model S.&#160;ratti, have an unusual life cycle that is advantageous for experimental genomic studies because it consists of both parasitic and free-living (environmental) generations3 (Figure 1). Specifically, both S.&#160;stercoralis and S.&#160;ratti can cycle through a single free-living generation. The free-living generation consists of post-parasitic larvae that develop into free-living adult males and females; all progeny of the free-living adults develop into infective larvae, which must infect a host to continue the life cycle. Furthermore, this environmental or free-living generation can be experimentally manipulated in the laboratory. Because free-living Strongyloides adults and C. elegans adults share similar morphology, techniques such as intragonadal microinjection that were originally developed for C. elegans can be adapted for use with free-living adult Strongyloides4,5. While DNA is generally introduced into free-living adult females, both males and females of Strongyloides can be microinjected6. Thus, functional genomic tools are available to interrogate many aspects of the biology of Strongyloides. Other parasitic nematodes lack a free-living generation, and as a result, are not as readily amenable to functional genomic techniques3.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63023/63023fig01.jpg" /> 
Figure 1: The Strongyloides stercoralis life cycle. The S.&#160;stercoralis parasitic females inhabit the small intestine of their mammalian hosts (humans, non-human primates, dogs). The parasitic females reproduce by parthenogenesis and lay eggs within the small intestine. The eggs hatch while still inside the host into post-parasitic larvae, which are then passed into the environment with feces. If the post-parasitic larvae are male, they develop into free-living adult males. If the post-parasitic larvae are female, they can either develop into free-living adult females (indirect development) or third-stage infective larvae (iL3s; direct development). The free-living males and females reproduce sexually to create progeny that are constrained to become iL3s. Under certain conditions, S.&#160;stercoralis can also undergo autoinfection, in which some of the post-parasitic larvae remain inside the host intestine rather than passing into the environment in feces. These larvae can develop into autoinfective larvae (L3a) inside the host, penetrate through the intestinal wall, migrate through the body, and eventually return to the intestine to become reproductive adults. The life cycle of S.&#160;ratti is similar, except that S.&#160;ratti infects rats and does not have an autoinfective cycle. The environmental generation is key to using Strongyloides species for genetic studies. The free-living adult females (P0) can be microinjected; their progeny, which will all become iL3s, are the potential F1 transgenics. This figure has been modified from Castelletto et al.3. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63023/63023fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
S.&#160;stercoralis shares many aspects of its biology with other gastrointestinal human-parasitic nematodes, including host invasion and host immune modulation. For example, human-parasitic hookworms in the genera Necator and Ancylostoma also infect by skin penetration, navigate similarly through the body, and ultimately reside as parasitic adults in the small intestine7. Thus, many gastrointestinal nematodes likely use common sensory behaviors and immune evasion techniques. As a result, the knowledge gleaned from Strongyloides will complement findings in other less genetically tractable nematodes and lead to a more complete understanding of these complex and important parasites.
This microinjection protocol outlines the method for introducing DNA into Strongyloides free-living adult females to make transgenic and mutant progeny. The strain maintenance requirements, including the developmental timing of adult worms for microinjections and the collection of transgenic progeny, are described. Protocols and a demonstration of the complete microinjection technique, along with protocols for culturing and screening transgenic progeny, are included, along with a list of all necessary equipment and consumables.

<table><tbody><tr><td>(&amp;minus;)-Nicotine, &amp;ge;99% (GC), liquid</td><td>Sigma-Aldrich</td><td>N3876-5ML</td><td>nicotine for paralyzing worms</td></tr><tr><td>3&quot; iron C-clamp, 3&quot; x 2&quot; (capacity x depth)</td><td>VWR</td><td>470121-790</td><td>C-clamp to secure setup to bench top</td></tr><tr><td>Agarose LE</td><td>Phenix</td><td>RBA-500</td><td>agarose for slides</td></tr><tr><td>Bone char, 4 lb pail, 10 x 28 mesh</td><td>Ebonex</td><td>n/a</td><td>charcoal for fecal-charcoal cultures</td></tr><tr><td>Bone char, granules, 10 x 28 mesh</td><td>Reade</td><td>bonechar10x28</td><td>charcoal for fecal-cultures (alternative to the above)</td></tr><tr><td>Coarse micromanipulator</td><td>Narishige</td><td>MMN-1</td><td>coarse micromanipulator</td></tr><tr><td>Corning Costar Spin-X centrifuge tube filters</td><td>Fisher</td><td>07-200-385</td><td>microfilter column</td></tr><tr><td>Cover glass, 48 x 60 mm, No. 1 thickness</td><td>Brain Research Lab</td><td>4860-1</td><td>coverslips (48 x 60 mm)</td></tr><tr><td>Deep Petri dishes, heavy version with 6 vents, 100 mm diameter</td><td>VWR</td><td>82050-918</td><td>10 cm Petri dishes (for fecal-charcoal cultures)</td></tr><tr><td>Eisco retort base w/ rod</td><td>Fisher</td><td>12-000-101</td><td>stand for Baermann apparatus</td></tr><tr><td>Eppendorf FemtoJet microinjector microloader tips</td><td>VWR</td><td>89009-310</td><td>for filling microinjection needles</td></tr><tr><td>Fisherbrand absorbent underpads</td><td>Fisher</td><td>14-206-62</td><td>bench paper (for prepping)</td></tr><tr><td>Fisherbrand Cast-Iron Rings</td><td>Fisher</td><td>14-050CQ</td><td>Baermann o-ring</td></tr><tr><td>Fisherbrand tri-cornered polypropylene beakers</td><td>Fisher</td><td>14-955-111F</td><td>Plastic beaker (for mixing)</td></tr><tr><td>Fisherbrand tri-cornered polypropylene beakers</td><td>Fisher</td><td>14-955-111F</td><td>Plastic beaker (for catch bucket/water bucket)</td></tr><tr><td>Fisherbrand tri-cornered polypropylene beakers</td><td>Fisher</td><td>14-955-111F</td><td>Plastic beaker (x2) (to make holder)</td></tr><tr><td>Gorilla epoxie in syringe</td><td>McMaster-Carr</td><td>7541A51</td><td>glue (to attach tubing)</td></tr><tr><td>Halocarbon oil 700</td><td>Sigma-Aldrich</td><td>H8898-50ML</td><td>halocarbon oil</td></tr><tr><td>High-temperature silicone rubber tubing for food and beverage, 1/2&quot; ID, 5/8&quot; OD</td><td>McMaster-Carr</td><td>3038K24</td><td>tubing (for funnel)</td></tr><tr><td>KIMAX funnels, long stem, 60&amp;deg; Angle, Kimble Chase</td><td>VWR</td><td>89001-414</td><td>Baermann funnel</td></tr><tr><td>Kimberly-Clark Professional Kimtech Science benchtop protectors</td><td>Fisher</td><td>15-235-101</td><td>bench paper (for prepping)</td></tr><tr><td>Leica stereomicroscope with fluorescence</td><td>Leica</td><td>M165 FC</td><td>GFP stereomicroscope for identifying and sorting transgenic worms</td></tr><tr><td>microINJECTOR brass straight arm needle-holder</td><td>Tritech</td><td>MINJ-4</td><td>microinjection needle holder</td></tr><tr><td>microINJECTOR system</td><td>Tritech</td><td>MINJ-1</td><td>microinjection system</td></tr><tr><td>Mongolian Gerbils</td><td>Charles River Laboratories</td><td>213-Mongolian Gerbil</td><td>gerbils for maintenance of S. stercoralis, male 4-6 weeks</td></tr><tr><td>Nasco Whirl-Pak easy-to-close bags, 18 oz</td><td>VWR</td><td>11216-776</td><td>Whirl-Pak sample bags</td></tr><tr><td>Nylon tulle (mesh)</td><td>Jo-Ann Fabrics</td><td>zprd_14061949a</td><td>nylon mesh for Baermann holder</td></tr><tr><td>Platinum wire, 36 Gauge, per inch</td><td>Thomas Scientific</td><td>1233S72</td><td>platinum/iridium wire for worm picks</td></tr><tr><td>Puritan tongue depressors, 152 mm (L) x 17.5 mm (W)</td><td>VWR</td><td>62505-007</td><td>wood sticks (for mixing samples)</td></tr><tr><td>QIAprep Spin Miniprep Kit (250)</td><td>QIAGEN</td><td>27106</td><td>QIAGEN miniprep kit</td></tr><tr><td>Rats-Long Evans</td><td>Envigo</td><td>140 HsdBlu:LE Long Evans</td><td>rats for maintenance of S. ratti, female 4-6 weeks</td></tr><tr><td>Rats-Sprague Dawley</td><td>Envigo</td><td>002 Hsd:Sprague Dawley SD</td><td>rats for maintenance of S. ratti, female 4-6 weeks</td></tr><tr><td>Really Useful Boxes translucent storage boxes with lids, 1.6 L capacity, 7-5/8&quot; x 5-5/16&quot; x 4-5/16&quot;</td><td>Office Depot</td><td>452369</td><td>plastic boxes for humidified chamber</td></tr><tr><td>Shepherd techboard, 8 x 16.5 inches</td><td>Newco</td><td>999589</td><td>techboard</td></tr><tr><td>Stainless steel raised wire floor</td><td>Ancare</td><td>R20SSRWF</td><td>wire cage bottoms</td></tr><tr><td>StalkMarket compostable cutlery spoons, 6&quot;, white, pack of 1,000</td><td>Office Depot</td><td>9587303</td><td>spoons</td></tr><tr><td>Stender dish, stacking type, 37 x 25 mm</td><td>Carolina (Science)</td><td>741012</td><td>watch glasses (small, round)</td></tr><tr><td>Stereomicroscope</td><td>Motic</td><td>K-400 LED</td><td>dissecting prep scope</td></tr><tr><td>Storage tote, color clear/white, outside height 4-7/8 in, outside length 13-5/8 in, Sterilite</td><td>Grainger</td><td>53GN16</td><td>plastic boxes for humidified chamber</td></tr><tr><td>Sutter P-30 micropipette puller</td><td>Sutter</td><td>P-30/P</td><td>needle puller with platinum/iridium filament</td></tr><tr><td>Syracuse watch glasses</td><td>Fisher</td><td>S34826</td><td>watch glasses (large, round)</td></tr><tr><td>Thermo Scientific Castaloy fixed-angle clamps</td><td>Fisher</td><td>05-769-2Q</td><td>funnel clamps (2x)</td></tr><tr><td>Three-axis hanging joystick oil hydrolic micromanipulator</td><td>Narishige</td><td>MM0-4</td><td>fine micromanipulator</td></tr><tr><td>United Mohr pinchcock clamps</td><td>Fisher</td><td>S99422</td><td>Pinch clamps (2x)</td></tr><tr><td>Vented, sharp-edge Petri dishes (60 mm diameter)</td><td>Tritech Research</td><td>T3308P</td><td>6 cm Petri dishes (for small-scale fecal-charcoal cultures)</td></tr><tr><td>VWR light-duty tissue wipers</td><td>VWR</td><td>82003-820</td><td>lining for Baermann holder</td></tr><tr><td>watch glass, square, 1-5/8 in</td><td>Carolina (Science)</td><td>742300</td><td>watch glasses (small, square)</td></tr><tr><td>Whatman qualitative grade plain circles, grade 1, 5.5 cm diameter</td><td>Fisher</td><td>09-805B</td><td>filter paper (for 6 cm Petri dishes)</td></tr><tr><td>Whatman qualitative grade plain circles, grade 1, 9 cm diameter</td><td>Fisher</td><td>09-805D</td><td>filter paper (for 10 cm Petri dishes)</td></tr><tr><td>World Precision Instrument borosilicate glass capillary, 1.2 mm x 4 in</td><td>Fisher</td><td>50-821-813</td><td>glass capillaries for microinjection needles</td></tr><tr><td>X-Acto Knives, No. 1 Knife With No. 11 Blade</td><td>Office Depot</td><td>238816</td><td>X-Acto knives without blades to hold worm picks</td></tr><tr><td>Zeiss AxioObserver A1</td><td>Zeiss</td><td>n/a</td><td>inverted microscope</td></tr></tbody></table>

generating transgenics and knockouts in strongyloides species by microinjection

The genus Strongyloides consists of multiple species of skin-penetrating nematodes with different host ranges, including Strongyloides stercoralis and Strongyloides ratti. S.&#160;stercoralis is a human-parasitic, skin-penetrating nematode that infects approximately 610 million people, while the rat parasite S.&#160;ratti is closely related to S.&#160;stercoralis and is often used as a laboratory model for S.&#160;stercoralis. Both S.&#160;stercoralis and S.&#160;ratti are easily amenable to the generation of transgenics and knockouts through the exogenous nucleic acid delivery technique of intragonadal microinjection, and as such, have emerged as model systems for other parasitic helminths that are not yet amenable to this technique.
Parasitic Strongyloides adults inhabit the small intestine of their host and release progeny into the environment via the feces. Once in the environment, the larvae develop into free-living adults, which live in feces and produce progeny that must find and invade a new host. This environmental generation is unique to the Strongyloides species and similar enough in morphology to the model free-living nematode Caenorhabditis elegans that techniques developed for C. elegans can be adapted for use with these parasitic nematodes, including intragonadal microinjection. Using intragonadal microinjection, a wide variety of transgenes can be introduced into Strongyloides. CRISPR/Cas9 components can also be microinjected to create mutant Strongyloides larvae. Here, the technique of intragonadal microinjection into Strongyloides, including the preparation of free-living adults, the injection procedure, and the selection of transgenic progeny, is described. Images of transgenic Strongyloides larvae created using CRISPR/Cas9 mutagenesis are included. The aim of this paper is to enable other researchers to use microinjection to create transgenic and mutant Strongyloides.

If the experiment was successful, the F1 larvae will express the transgene and/or mutant phenotype of interest (Figure 4). However, transformation rates are highly variable and depend on the constructs, the health of the worms, the post-injection culturing conditions, and the skill of the experimenter. In general, a successful experiment will yield &#62;15 F1 larvae per injected female and a transformation rate of &#62;3% for fluorescent markers. If the total number of living progeny averages to fewer than 10 larvae/female, then it is possible that the construct is toxic, and the transformed larvae are not surviving. Finding large numbers of fluorescent eggs but not fluorescent larvae is another indication that the injection mix may be toxic. When first learning the technique, it is recommended to use a construct that expresses well, such as act-2::mRFPmars, which drives robust expression in body wall muscle13 (Figure 4).
When generating mutants by CRISPR/Cas9-mediated targeted mutagenesis, the use of a repair template containing an act-2::mRFPmars or act-2::GFP transgene13 is recommended so that potential mutants can be identified based on fluorescence9,14,15. It is important to note that because Strongyloides express transgenes from extrachromosomal arrays, fluorescent F1 progeny may express mRFPmars or GFP from the array alone or express mRFPmars or GFP from both the array and an integrated transgene3,9,16. It is possible to identify larvae that are more likely to have integrated transgenes based on the pattern of fluorescent expression: &#34;patchy&#34; expression in the body wall muscle (Figure 4A) is more common when the transgene is not integrated into the genome, whereas consistent expression throughout the body wall muscle (Figure 4B) often, but not always, indicates that the transgene has integrated into the genome. However, expression patterns alone cannot be used to conclusively identify mutants-some worms with consistent expression throughout the body wall muscle may not have integration events. Moreover, expression patterns cannot distinguish mutants that are homozygous from those that are heterozygous or mosaic. Thus, each worm must be PCR-genotyped9,14,15. When disrupting genes that yield easily visible phenotypes, it may not be necessary to use a repair template. For example, disruption of the Strongyloides unc-22 gene results in a dominant &#34;twitcher&#34; phenotype, with rates of heterozygous or homozygous disruptions above 10%9.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63023/63023fig04.jpg" /> 
Figure 4: Transgenic Strongyloides stercoralis larvae. (A, B)&#160;S.&#160;stercoralis larvae expressing an act-2::mRFPmars transgene, which expresses in the body wall muscle13. The transgene was incorporated into a repair template for CRISPR/Cas9-mediated disruption of the Ss-unc-22 locus9. (A) An S.&#160;stercoralis larva with an incomplete, or &#34;patchy,&#34; act-2::mRFPmars expression pattern that may indicate expression from an extrachromosomal array. (B) An S.&#160;stercoralis iL3 expressing the more complete act-2::mRFPmars expression pattern that may indicate gene disruption and integration of the repair template. For A, B, panels show differential interference contrast (left), fluorescent (middle), and merged (right) images. Scale bars = 50 &#956;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63023/63023fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1: Demonstration of the needle-breaking process to make a useable needle. The tip of the needle is tapped against the edge of a glass shard (object on far left). When liquid emerges, the needle is pulled back and moved down onto the agar. The needle tip comes to a sharp point, and liquid flows moderately fast. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63023/Movie 1 2021-07-22.mov" target="_blank">Please click here to download this Video.</a>
Video 2: Demonstration of a successful injection. The posterior arm of the gonad is visible as a light gray structure on the right. The tip of the needle and the gonad must be in the same focal plane. If the needle slides over or under the worm or along the body without catching, adjust the position. The tip of the needle will slightly indent the body wall. A quick tap on the needle holder attached to the micromanipulator will gently push the tip through the body wall and into the gonad. Once the needle is inside the gonad, apply pressure and inject the DNA solution. The liquid should visibly flood the gonad. If the wound closes when the tip of the needle is removed, the worm is likely to survive. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63023/Movie 2.mov" target="_blank">Please click here to download this Video.</a>
Supplemental Material: Strongyloides strain maintenance. This protocol outlines the maintenance procedure for S.&#160;stercoralis in gerbils and S.&#160;ratti in rats. It includes the infective dose used for each nematode and host. Hosts are infected via subcutaneous injections under anesthesia. The progression of infections from patency to peak larval output to loss of patency is described for each nematode-host combination. The protocol used to collect infested feces and make the fecal-charcoal cultivation plates is also described. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63023/Castelletto_and_Hallem 2021 supp 2021-08-30.docx" target="_blank">Please click here to download this File.</a>
Supplemental Figure S1: An image of a microinjection slide consisting of a dried agar pad on a coverslip with a small glass shard for breaking the microinjection needle. The agar outline and the shard outline are added here for clarity. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63023/Figure_S1_2021-7-29.pdf" target="_blank">Please click here to download this File.</a>
Supplemental Movie S1: Demonstration of an injection resulting in a damaged gonad. The anterior arm of the gonad is in focus. Applying slight pressure shows that the solution in the needle is still flowing out freely. The tip of the needle is in the same focal plane as the gonad and is aimed at a slight angle. Once the tip of the needle is inserted, the solution is injected into the worm and fills the gonad. However, when the needle is removed, a piece of the gonad protrudes through the wound in the cuticle. Material is visibly flowing out. This worm is unlikely to survive. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63023/Movie S1 2021-07-31v2.mp4" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Generating Transgenics and Knockouts in Strongyloides Species by Microinjection at JoVE.com

Generating Transgenics and Knockouts in Strongyloides Species by Microinjection

The functional genomic toolkit for the parasitic nematodes Strongyloides stercoralis and Strongyloides ratti includes transgenesis, CRISPR/Cas9-mediated mutagenesis, and RNAi. This protocol will demonstrate how to use intragonadal microinjection to introduce transgenes and CRISPR components into S.&#160;stercoralis and S.&#160;ratti.

Generating Transgenics and Knockouts in Strongyloides Species by Microinjection

The genus Strongyloides consists of multiple species of skin-penetrating nematodes with different host ranges, including Strongyloides stercoralis and ...

generating-transgenics-knockouts-strongyloides-species

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

2.6K Views.  University of California, Los Angeles. The functional genomic toolkit for the parasitic nematodes Strongyloides stercoralis and Strongyloides ratti includes transgenesis, CRISPR/Cas9-mediated mutagenesis, and RNAi. This protocol will demonstrate how to use intragonadal microinjection to introduce transgenes and CRISPR components into S. stercoralis and S. ratti.

Video: Generating Transgenics and Knockouts in Strongyloides Species by Microinjection

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. ...

Genetics

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 ...

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Biology

Generation of Genetically Modified Mice through the Microinjection of Oocytes

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear injection of DNA expression constructs into fertilized oocytes remains the most commonly used technique to generate transgenic mice for overexpression. With the introduction of CRISPR technology for gene targeting, pronuclear injection into fertilized oocytes has been extended to the generation of both knockout and knockin mice. This work describes the preparation of DNA for injection and the generation of CRISPR guides for gene targeting, with a particular emphasis on quality control. The genotyping procedures required for the identification of potential founders are critical. Innovative genotyping strategies that take advantage of the "multiplexing" capabilities of CRISPR are presented herein. Surgical procedures are also outlined. Together, the steps of the protocol will allow for the generation of genetically modified mice and for the subsequent establishment of mouse colonies for a plethora of research fields, including immunology, neuroscience, cancer, physiology, development, and others.

The microinjection of mouse oocytes is commonly used for both classic transgenesis (i.e., the random integration of transgenes) and CRISPR-mediated gene targeting. This protocol reviews the latest developments in microinjection, with a particular emphasis on quality control and genotyping strategies.

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

Developmental Biology

phiC31 Integrase-Mediated Embryo Transgenesis in Drosophila

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

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 ...

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily produced by electroporation of CRISPR factors (and single-stranded oligo DNA donors) into the zygote. In contrast, gene cassette (&#62;1 kb) knock-in and floxed mice are mainly generated by microinjection of CRISPR factors and double-stranded DNA donors into zygotes. Genome editing technologies have also increased the flexibility of genetically modified mice production. It is now possible to introduce the intended mutations in the target genomic regions in a number of beneficial inbred mouse strains. Our team has produced over 200 gene cassette knock-in mouse lines, and over 110 floxed mouse lines by zygote microinjection of CRISPR-Cas9 following requests from several countries, including Japan. Some of these genome editing used BALB/c, C3H/HeJ, and C57BL/6N inbred strains, however most used C57BL/6J. Unlike the electroporation method, genome editing by zygote microinjection in various inbred strains of mice is not that easy. However, gene cassette knock-in and floxed mice on single inbred genetic backgrounds are as critical as genetic humanized, fluorescent reporter, and conditional knockout mouse models. Therefore, this article presents the protocol for the zygote microinjection of CRISPR factors and double-stranded DNA donors in C57BL/6J mice for generating gene cassette knock-in and floxed mice. This article exclusively focuses on nuclear injection rather than cytoplasmic injection. In addition to zygote microinjection, we outline the timeline for the production process and peripheral techniques such as induction of superovulation and embryo transfer.

The present protocol describes zygote microinjection of CRISPR-Cas9 and donor DNA to efficiently produce gene cassette knock-in and floxed mice.

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily ...

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans3, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4 and Pristionchus pacificus5. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior6-7, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4 and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F1 progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

A Method for Microinjection of Patiria minata Zygotes

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and evolution of developmental processes. The sea star, Patiria miniata, is gaining prevalence as a model system for these types of studies which were previously performed almost exclusively in the sea urchins, Strongylocentrotus purpuratus and Lytechinus variegatus. An advantage of these model systems is the ease of producing modified embryos in which a particular gene is up or downregulated, labeling a group of cells, or introducing a reporter gene. A single microinjection method is capable of creating a wide variety of such modified embryos. Here, we present a method for obtaining gametes from P. miniata, producing zygotes, and introducing perturbing reagents via microinjection. Healthy morphant embryos are subsequently isolated for quantitative and qualitative studies of gene function. The availability of genome and transcriptome data for this organism has increased the types of studies that are performed and the ease of executing them.

A Method for Microinjection of Patiria minata Zygotes

Methods that produce morphant embryos are essential to study developmental mechanisms and gene regulatory networks. The sea star Patiria miniata is an emerging model system for these studies. Here we present a protocol for obtaining gametes, producing cultures of embryos, and rapid microinjection of zygotes from this species.

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and ...

Gonad Microinjection: A Method of Compound Delivery Directly into the Germline of C. elegans

Source: Farboud, B. et al, <a href="https://www-jove-com.bdigitaluss.remotexs.co/video/57350/enhanced-genome-editing-with-cas9-ribonucleoprotein-diverse-cells">Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms.</a>&#160;J. Vis. Exp. (2018).
This video describes microinjection, a common method of creating transgenic C. elegans strains.&#160; In the example, we will see microinjection used to introduce a ribonucleoprotein (RNP) complex for CRISPR-bases gene editing.

Mikroiniekcja gonad: metoda dostarczania związków bezpośrednio do linii rozrodczej C. elegans

Source: Farboud, B. et al, Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms.&#160;J. Vis. Exp. (2018).
This video ...

Generating Transgenics and Knockouts in Strongyloides Species by Microinjection

Podsumowanie

Przeglądaj więcej filmów

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

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

Generation of Stable Transgenic C. elegans Using Microinjection

Generation of Genetically Modified Mice through the Microinjection of Oocytes

Embryo Microinjection for Transgenesis in Drosophila

Generation of Transgenic C. elegans by Biolistic Transformation

Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

A Method for Microinjection of Patiria minata Zygotes

Mikroiniekcja gonad: metoda dostarczania związków bezpośrednio do linii rozrodczej C. elegans