We thank the KYOTO Drosophila Stock Center for fly strains. We also thank Ms. Wanda Miyata for English language editing of the manuscript and Dr. Jeremy Allen from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript. This work was supported by grants (JP16km0210072, JP17km0210146, JP18km0210146) from the Japan Agency for Medical Research and Development (AMED) to T.T.-S.-K., grants (JP16km0210073, JP17km0210147, JP18km0210145) from AMED to S.K., a grant (JP20km0210172) from AMED to T.T.-S.-K. and S.K., a Grant-in-Aid for Scientific Research (C) (JP19K06780) from the Japan Society for the Promotion of Science (JSPS) to T.T.-S.-K., and a Grant-in-Aid for Scientific Research on Innovative Areas (JP18H05552) from JSPS&#160;to S.K.

1. Equipment preparation
<ol>
	<li>Micromanipulator system: Assemble a micromanipulator system to collect and transplant cells (Figure 1A).</li>
	<li>PGC-collection glass slides (Figure 2A)
	<ol>
		<li>To prepare heptane glue, cut approximately 30 cm long double-sided tape and soak it overnight in 7 mL of technical (regular)-grade heptane solution.</li>
		<li>Draw two parallel reference lines for embryo alignment on the back of a glass slide.</li>
		<li>Spread drops of the above heptane glue on the glass slide (on the side without the lines) using a Pasteur pipette. Air-dry the surface of the slide until it becomes white.</li>
		<li>Repeat the addition and spreading of heptane-glue drops and dry the slide again. 
		NOTE: The glue prevents liquid solutions from spreading over the flat surface and makes it easier to load aqueous solutions into a needle.</li>
		<li>To make embryo-pool frames, stick three layers of 0.2 mm-thick standard vinyl tape, such as electrical tape, on a cutting board. Cut the tape into 1.5 cm-wide rectangles. Then, cut away all three layers of tape, leaving a 2 to 3 mm frame. 
		NOTE: An embryo-pool frame is affixed after aligning embryos, to form a pool for embryos.</li>
	</ol>
	</li>
	<li>Transplantation needles 
	NOTE: All commercially available needles at the time of this study were too narrow or too broad for PGC cryopreservation.
	<ol>
		<li>Make a needle using a glass capillary and a puller. We use a NARISHIGE&#160;PN-31 puller with the heater level at 85.0-98.4, the magnet main level at 57.8, and the magnet sub-level at 45.0.</li>
		<li>To make a needle with an approximate wall thickness of 1 &#181;m and a tip of approximately 200 &#181;m in length with an inner diameter of 10-12 &#181;m, polish the needle tip in the following three-step process (Figure 3). First, grind down the needle tip at an angle of 30&#176; at a speed of 780 rpm until the tip has an inner diameter of 10-12 &#181;m. This first grinding step takes approximately 1 h. 
		NOTE: To avoid breaking the needle tip, first rotate the grindstone and then gently move the needle down onto the grindstone.</li>
		<li>Draw a line on the top of the needle to track the desired angle. Rotate the needle counterclockwise at 90&#176; and polish it again at a speed of 180 rpm. This takes approximately 5 min.</li>
		<li>Rotate the needle clockwise 45&#176; and polish it at a speed of 180 rpm for one second.</li>
		<li>Place a collection glass slide with a drop of chromic acid mixture (CAUTION: toxic) onto the microscope stage. Attach the needle to the capillary holder (Figure 1D) at a 10&#176;-13&#176; angle relative to the slide surface, carefully move the needle down, and immerse the tip in the chromic acid mixture.</li>
		<li>By pulling and pushing the plunger (Figure 1B), mechanically load and discharge the solution from the needle several times to remove glass debris in the needle. Be sure to clean the outer wall as well.</li>
		<li>Wash the inside and outside of the needle two times&#160;with distilled water to remove the chromic acid completely.</li>
	</ol>
	</li>
</ol>
2. Collection and cryopreservation of PGCs
<ol>
	<li>Collecting embryos
	<ol>
		<li>Transfer an appropriate number of flies of the donor strain of interest (approximately 450 for each sex for the embryo-collection cup) into an embryo-collection cup with an embryo-collection plate (Figure 1E) and incubate them at 25 &#176;C. We usually use 3- to 5-day-old parental flies that are reared under less crowded conditions at room temperature (23-25 &#176;C).</li>
		<li>Perform two 30-min pre-collections and discard any eggs laid. Because females can retain fertilized eggs that develop in the oviduct, this step is required to synchronize egg laying in step 2.1.3 (Figure 4).</li>
		<li>After the two pre-collections, collect embryos for 50 min and then incubate the collected embryos in a humidified chamber at 25 &#176;C to allow the embryos to develop to the blastoderm stage (early stage 517). The incubation time is usually 100 min but can be extended up to 120 min, depending on the strain (Figure 4). 
		NOTE: A humidified chamber is made by placing a moist paper towel at the bottom of a plastic box and spraying it with a mist of water before use. In early stage-5 embryos, PGC formation is complete, but somatic cellularization is not. The exact stage of an embryo is determined under a compound microscope in step 2.4.</li>
	</ol>
	</li>
	<li>Dechorionating embryos
	<ol>
		<li>Deposit a drop of distilled water on a stainless-steel mesh strainer (150 mesh, 109 &#181;m opening, 60 &#181;m wire diameter; Figure 1F). Using forceps, collect embryos from the embryo-collection plate and put them in the water droplet.</li>
		<li>Press tissue paper against the strainer from underneath to absorb the water. Add droplets of fresh 5% (as Cl) sodium hypochlorite solution to the embryos and continuously tap the strainer for 10 s.</li>
		<li>Wash the embryos by directly splashing them with distilled water and press tissue paper against the strainer from underneath to absorb the water. Repeat this step 3x.</li>
	</ol>
	</li>
	<li>Aligning dechorionated embryos
	<ol>
		<li>Under a stereo microscope, use forceps to transfer embryos. Align the dechorionated embryos in two rows on a PGC-collection glass slide along the two reference lines (Figure 2A). The embryos are oriented with their anterior to the right (the side to be manipulated) and ventral side up. 
		NOTE: This step should be finished in 20 min, during which we usually align approximately 40 embryos.</li>
		<li>Affix an embryo-pool frame around the embryos on the PGC-collection glass slide. Drop 1 &#181;L of CPA solution (1x Ephrussi-Beadle Ringer solution, EBR, containing 20% ethylene glycol and 1 M sucrose; 1x EBR: 130 mM NaCl, 5 mM KCl, 2 mM CaCl2, and 10 mM Hepes at pH 6.9) at two separate spots in the area enclosed by the frame and fill the pool with silicone oil to prevent the embryos from drying out (Figure 2A). 
		NOTE: To prepare the CPA solution, completely dissolve 10.26 g of sucrose in approximately 20 mL of distilled H2O containing 3 mL of 10 x EBR solution. Add 6 mL of ethylene glycol and then add distilled H2O up to 30 mL. After thorough mixing, filter the solution through a 0.22 mm disposable membrane.</li>
	</ol>
	</li>
	<li>Collecting PGCs
	<ol>
		<li>Place the PGC-collection glass slide of step 2.3.2 onto the stage of a microscope equipped with a micromanipulator system. Attach the needle to the capillary holder and bring the first embryo in the left row and the needle tip into the same focal plane. Load silicone oil into the needle for 2-3 s.</li>
		<li>Begin PGC collection from embryos in the left row. Using a 20x objective lens, gently move the needle tip to the surface of the anterior end of the embryo and penetrate the embryo toward the posterior end, not by moving the needle but by moving the microscope stage.</li>
		<li>When the needle tip reaches the posterior end, retract the needle slightly and completely discharge any yolk in the needle just inside the somatic cell layer.</li>
		<li>While keeping the pressure in the needle constant, move the needle tip to the PGCs just inside the posterior pole and gently, but without taking much time, load the PGCs.</li>
		<li>Pull the needle out of the embryo quickly and discharge yolk and other contaminants from the needle into the silicone oil pool, keeping the PGCs in the needle. Then, load clean silicone oil from the pool.</li>
		<li>Repeat steps 2.4.2 to 2.4.5 for the other embryos in the left row. Before collecting PGCs from a new embryo, deposit as much of the silicone oil loaded in step 2.4.5 inside the somatic cell layer as possible while keeping loaded PGCs in the needle. This ensures that newly loaded PGCs are adjacent to the previously collected PGCs without any intervening material between them.</li>
		<li>After completing PGC collection from embryos in the left row, separate PGCs from the yolk and other contaminants as much as possible. To achieve this, deposit all PGCs in the needle onto the surface of an embryo and remove any yolk or other contaminants to another neighboring embryo.</li>
		<li>Next, collect PGCs from embryos in the right row. Combine the PGCs collected from the right and left rows.</li>
	</ol>
	</li>
	<li>Applying cryoprotective agent (CPA) to PGCs
	<ol>
		<li>After washing the needle with CPA in one drop, load fresh CPA in&#160;another drop into the needle and add CPA to the PGCs deposited on the embryo. The volume of CPA should be equivalent to that of the PGCs.</li>
		<li>Remove as much CPA as possible from the cluster of PGCs 1-2 s after the addition of CPA. PGCs shrink slightly and become square in shape immediately after CPA addition.</li>
		<li>Empty the needle and then load silicone oil for 5 s or longer. Load all the collected PGCs and then load silicone oil once again for 5 s or longer. PGCs are now sandwiched between two layers of silicone oil (Figure 2B). 
		NOTE: It is important to remove as much yolk, CPA, and other contaminants as possible.</li>
	</ol>
	</li>
	<li>Cryopreserving PGCs
	<ol>
		<li>Open the three-way stopcock (Figure 1C) and then detach the needle from the micromanipulator. Blot the oil off the surface of the needle with soft tissue paper. Do not directly touch the tip of the needle with the tissue.</li>
		<li>Attach the needle to a needle holder and lock it in position at the base using vinyl tape (Figure 1H). Affix a label to the holder tube.</li>
		<li>Flash freeze the holder with the needle pointing downwards by submerging it in liquid nitrogen. Do not release the holder until the liquid stops fizzing out of the rack.</li>
		<li>Store the holder in a liquid nitrogen storage tank in the liquid phase area, not the vapor phase area.</li>
	</ol>
	</li>
</ol>
3. Thawing and transplanting PGCs
<ol>
	<li>Collecting, dechorionating, and aligning embryos from agametic host flies
	<ol>
		<li>Collect and dechorionate embryos from agametic host flies following step 2.</li>
		<li>Align the stage 5 agametic host embryos on a transplantation glass slide. However, this time, orient the posterior to the right (the side to be manipulated) and ventral to the top (Figure 5). Line up approximately 30 embryos in two rows in 20 min.</li>
		<li>While aligning embryos, operate a humidifier for 2-10 min if the room humidity requires it (Table 1). The ideal humidity is 30% to 40%, but this can vary depending on thermal conditions.</li>
	</ol>
	</li>
	<li>Thawing and transplanting PGCs in host embryos
	<ol>
		<li>To quickly thaw cryopreserved PGCs, slip the holder containing the needle into room temperature 1x EBR solution with the needle pointing downwards and keep it submerged for 10 s.</li>
		<li>Place the transplantation glass slide on the stage of a microscope. Attach the freeze-thawed needle to the capillary holder and bring the first embryo in the left row and the needle tip into the same focal plane.</li>
		<li>Using a 20x objective lens, gently move the needle tip to the surface of the posterior end of the embryo.</li>
		<li>Gently prod the outside of each embryo and make sure that they slowly return to their original shape. The prodding will confirm that the inner&#160;pressure of the embryo is not too high or too low.&#160;</li>
		<li>Gently move the needle and penetrate an embryo from the posterior pole.</li>
		<li>Gently deposit approximately 10-20 PGCs just inside the posterior pole, precisely between the vitelline membrane and the somatic cell layer of the embryo. Avoid depositing them in the somatic cell layer. If the perivitelline fluid leaks out of the embryo, suck the leaked fluid into the needle and remove it.</li>
		<li>Retract the needle from the embryo. Repeat steps 3.2.5 and 3.2.6 for subsequent embryos.</li>
	</ol>
	</li>
</ol>
4. Incubating embryos and restoring donor strains
<ol>
	<li>Remove any embryos that do not receive transplanted PGCs and incubate the remaining embryos in a humidified chamber (Figure 1G) at 25 &#176;C.</li>
	<li>At 24 h or more after transplantation and as soon as possible after hatching, use forceps to pick up and transfer hatched larvae to standard Drosophila food vials and incubate at 25 &#176;C.</li>
	<li>To revive the strain, cross newly emerged females and males (Figure 6). 
	&#8203;NOTE: Agametic hosts make it possible to restore the entire genome at once without crossing to balancer-chromosome strains. The co-existence of agametic males in a vial will not matter because females, even if mated with them, do not show long-term post-mating responses, including decreased receptivity to remating18,19.</li>
</ol>

Cryopreservation of PGCs for Long-Term Preservation of Drosophila Strains

<ol>
	<li>Br&#252;schweiler, W., Gehring, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+freezing+living+ovaries+of+Drosophila+melanogaster+larvae+and+its+application+to+the+storage+of+mutant+stocks.">A method for freezing living ovaries of Drosophila melanogaster larvae and its application to the storage of mutant stocks.</a> Experientia. 29, 134-135 (1973).</li><li>Steponkus, P. L., 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=Cryopreservation+of+Drosophila+melanogaster+embryos.">Cryopreservation of Drosophila melanogaster embryos.</a> Nature. 345, 170-172 (1990).</li><li>Mazur, P., Cole, K. W., Hall, J. W., Schreuders, P. D., Mahowald, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryobiological+preservation+of+Drosophila+embryos.">Cryobiological preservation of Drosophila embryos.</a> Science. 258 (5090), 1932-1935 (1992).</li><li>Zhan, L., Li, M. G., Hays, T., Bischof, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+method+for+Drosophila+melanogaster+embryos.">Cryopreservation method for Drosophila melanogaster embryos.</a> Nat Comm. 12, 2412 (2021).</li><li>Van Deusen, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+determination+in+germ+line+chimeras+of+Drosophila+melanogaster.">Sex determination in germ line chimeras of Drosophila melanogaster.</a> Development. 37 (1), 173-185 (1977).</li><li>Breen, T. R., Duncan, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+expression+of+genes+that+regulate+the+bithorax+complex+of+Drosophila+melanogaster.">Maternal expression of genes that regulate the bithorax complex of Drosophila melanogaster.</a> Dev Biol. 118, 442-456 (1986).</li><li>Schupbach, T., Wieschaus, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+autonomy+of+maternal-effect+mutations+altering+the+embryonic+body+pattern+of+Drosophila.">Germline autonomy of maternal-effect mutations altering the embryonic body pattern of Drosophila.</a> Dev Biol. 113, 443-448 (1986).</li><li>Irish, V., Lehmann, R., Akam, 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+posterior-group+gene+nanos+functions+by+repressing+hunchback+activity.">The Drosophila posterior-group gene nanos functions by repressing hunchback activity.</a> Nature. 338, 646-648 (1989).</li><li>H&#252;lskamp, M., Schr&#246;der, C., Pfeifle, C., J&#228;ckle, H., Tautz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posterior+segmentation+of+the+Drosophila+embryo+in+the+absence+of+a+maternal+posterior+organizer+gene.">Posterior segmentation of the Drosophila embryo in the absence of a maternal posterior organizer gene.</a> Nature. 338, 629-632 (1989).</li><li>Steinmann-Zwicky, M., Schmid, H., N&#246;thiger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-autonomous+and+inductive+signals+can+determine+the+sex+of+the+germ+line+of+Drosophila+by+regulating+the+gene+Sxl.">Cell-autonomous and inductive signals can determine the sex of the germ line of Drosophila by regulating the gene Sxl.</a> Cell. 57 (1), 157-166 (1989).</li><li>Stein, D., Roth, S., Vogelsang, E., N&#252;sslein-Volhard, 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+polarity+of+the+dorsoventral+axis+in+the+drosophila+embryo+is+defined+by+an+extracellular+signal.">The polarity of the dorsoventral axis in the drosophila embryo is defined by an extracellular signal.</a> Cell. 65 (5), 725-735 (1991).</li><li>Kobayashi, S., Yamada, M., Asaoka, M., Kitamura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+role+of+the+posterior+morphogen+nanos+for+germline+development+in+Drosophila.">Essential role of the posterior morphogen nanos for germline development in Drosophila.</a> Nature. 380, 708-711 (1996).</li><li>Asaoka, 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=Offspring+production+from+cryopreserved+primordial+germ+cells+in+Drosophila.">Offspring production from cryopreserved primordial germ cells in Drosophila.</a> Comm Biol. 4 (1), 1159 (2021).</li><li>Blitz, I. L., Fish, M. B., Cho, K. W. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leapfrogging:+primordial+germ+cell+transplantation+permits+recovery+of+CRISPR/Cas9-induced+mutations+in+essential+genes.">Leapfrogging: primordial germ cell transplantation permits recovery of CRISPR/Cas9-induced mutations in essential genes.</a> Development. 143 (15), 2868-2875 (2016).</li><li>Koslov&#225;, A., 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=Precise+CRISPR/Cas9+editing+of+the+NHE1+gene+renders+chickens+resistant+to+the+J+subgroup+of+avian+leukosis+virus.">Precise CRISPR/Cas9 editing of the NHE1 gene renders chickens resistant to the J subgroup of avian leukosis virus.</a> Proc Natl Acad Sci U S A. 117 (4), 2108-2112 (2020).</li><li>Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+generation+of+zebrafish+maternal-zygotic+mutants+through+transplantation+of+ectopically+induced+and+Cas9/gRNA+targeted+primordial+germ+cells.">Efficient generation of zebrafish maternal-zygotic mutants through transplantation of ectopically induced and Cas9/gRNA targeted primordial germ cells.</a> J Genet Genom. 47 (1), 37-47 (2020).</li><li>Campos-Ortega, J. A., Hartenstein, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+Drosophila+Embryogenesis.">Stages of Drosophila Embryogenesis.</a> The Embryonic Development of Drosophila. , (1997).</li><li>Manning, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sperm+factor+affecting+the+receptivity+of+Drosophila+melanogaster+females.">A sperm factor affecting the receptivity of Drosophila melanogaster females.</a> Nature. 194, 252-253 (1962).</li><li>Kubli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex-peptides:+seminal+peptides+of+the+Drosophila+male.">Sex-peptides: seminal peptides of the Drosophila male.</a> Cell Mol Life Sci. 60, 1689-1704 (2003).</li><li>Lehmann, R., N&#252;sslein-Volhard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abdominal+segmentation,+pole+cell+formation,+and+embryonic+polarity+require+the+localized+activity+of+oskar,+a+maternal+gene+in+drosophila.">Abdominal segmentation, pole cell formation, and embryonic polarity require the localized activity of oskar, a maternal gene in drosophila.</a> Cell. 47 (1), 141-152 (1986).</li><li>Kiger, A. A., Gigliotti, S., Fuller, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+genetics+of+the+essential+Drosophila+Nucleoporin+nup154:+allelic+differences+due+to+an+outward-directed+promoter+in+the+P-element+3&#8242;+end.">Developmental genetics of the essential Drosophila Nucleoporin nup154: allelic differences due to an outward-directed promoter in the P-element 3&#8242; end.</a> Genetics. 153 (2), 799-812 (1999).</li><li>Rienzi, L. F., 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=Perspectives+in+gamete+and+embryo+cryopreservation.">Perspectives in gamete and embryo cryopreservation.</a> Semin Reprod Med. 36 (5), 253-264 (2018).</li></ol>

The authors have no conflicts of interest to declare.

A critical factor for success in PGC cryopreservation and revival is to use good embryos. Young females (e.g., 3- to 5-day-old) should be used for embryo collection. Both donor and host embryos are assessed by microscopic inspection, and only those at the blastoderm stage (stage 5) are used12. For PGC collection, we usually align approximately 40 donor embryos in a 20 min period and collect PGCs from approximately 30 embryos at early stage 5; older and defective embryos are not used. After cryopreservation and thawing, PGCs should maintain their shape; PGCs rupture in unsuccessful preservation. Host embryos should also be at stage 5 and have a moderate inner&#160;pressure; embryos should slowly return to their original shape after gentle prodding. Overly and insufficiently dried embryos will not develop normally after transplantation. Because heterosexual transplantation of PGCs fails to produce gametes in Drosophila5,10, transplantation of PGCs from multiple donor embryos into host embryos is more likely to yield fertile adults. To this end, we usually collect PGCs from approximately 30 embryos per needle.
As cryoprotectants, we tried ethylene glycol, dimethyl sulfoxide, and glycerol together with sucrose at various concentrations. We determined EBR containing 20% ethylene glycol and 1 M sucrose to be the best13; however, the use of different cryoprotectants may improve PGC preservation22.
This cryopreservation method requires specialized skills in PGC handling, and approximately 6 weeks of training is needed to comfortably collect and transplant PGCs. To assess and improve skill proficiency, this may be broken into six training steps: 1) aligning embryos on a glass slide, 2) controlling a manipulator, 3) transplanting PGCs from an embryo into another embryo without cryopreservation, 4) transplanting PGCs from 10 or more embryos into 5 to 10 embryos, 5) transplanting PGCs after applying CPA, and 6) transplanting PGCs after freeze-thawing. Each step may take 1 week. The short-term goals at step 3 are a hatching rate of 40%, embryo-to-adult viability of 10%-20%, and a frequency of fertile flies of 20%.
PGC cryopreservation requires costly instrumentation and highly skilled personnel. Therefore, this method may not be adopted by many laboratories. However, the current PGC method has several important aspects. First, PGCs are much smaller than embryos and are very permeable to cryoprotectants. In contrast, cryoprotectant permeability is severely limited by the waxy layers of Drosophila embryos, which is the most serious problem in embryo cryopreservation. Indeed, previous studies have made great efforts to find a time window in which embryos have a high survival rate and a thinner wax layer. The second is concerned with developmental and morphological variation among strains. PGCs are collected from early stage-5 embryos (2 h 30 min-3 h 20 min after egg laying), while embryo cryopreservation is performed on stage-16 embryos (14-22 h after egg laying). The embryos are, therefore, much older and show much larger strain variation in the optimal time window for cryopreservation compared with PGC cryopreservation. Indeed, the frequency of hosts producing donor-derived progeny did not vary among five strains studied by Asaoka et al.13, although the hosts were not agametic. Moreover, PGCs have the potential to be used in genetic engineering applications, such as genome editing14,15,16.

The maintenance of Drosophila strains by the transfer of adult flies to new food vials inevitably results in the accumulation of mutations and epigenetic changes over time. Development of an alternative method for long-term maintenance of Drosophila strains without such changes is imperative, especially for reference strains in which the whole genome has to be maintained. Several successful attempts to cryopreserve Drosophila embryos or ovaries have been described1,2,3. Unfortunately, they are still not of practical use because of low reproducibility. Indeed, early-stage embryos have a low survival rate after cryopreservation because of their high yolk content, which impedes cryoprotective agent (CPA) permeation and diffusion2,3. CPA permeability is also severely limited by the waxy layers of late-stage embryos. It is difficult and time-consuming to find a strain-specific time period in which embryos have a high survival rate and a thinner wax layer. Recently, Zhan et al.4 improved methods for embryo permeabilization, CPA loading, and vitrification and successfully cryopreserved embryos of multiple strains. However, the methods are not easy to apply because the viability of embryos after permeabilization tends to be poor. Therefore, further improvement and development of alternative approaches are still needed. Methods involving the cryopreservation of primordial germ cells (PGCs) are an alternative approach for the long-term maintenance of Drosophila strains.
PGC (also called pole cell) transplantation has been used to generate germline chimeras, especially females, to study processes such as maternal effects of zygotic lethal mutations and sex determination of germ cells5,6,7,8,9,10,11,12. PGCs are much smaller than embryos and are likely to be highly permeable to most cryoprotectants. Furthermore, developmental and morphological variation among strains is less problematic, and an agametic host enables quick restoration of whole genomes. We recently developed a new method of PGC cryopreservation13, which prevents the otherwise inevitable genetic and epigenetic changes in Drosophila strains. Here, we present the detailed protocol.
This cryopreservation method requires specific expertise in PGC handling and instrumentation. While a step-by-step approach may be an efficient solution for those who are unfamiliar with it, it may be unsuitable for small laboratories due to instrumentation requirements. This PGC cryopreservation protocol can be more easily adapted for use with different Drosophila species and different insect species than embryo cryopreservation protocols because of smaller developmental and morphological differences. PGCs can also potentially be used in genetic engineering applications, such as genome editing14,15,16. In summary, this method can be used in stock centers and other laboratories to maintain fly and other insect strains for prolonged periods of time without changes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetic acid</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>017-00256</td>
			<td>For embryo collection</td>
		</tr>
		<tr>
			<td>Agar powder</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>010-08725</td>
			<td>For embryo collection</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>038-24985</td>
			<td>For EBR solution</td>
		</tr>
		<tr>
			<td>Capillary</td>
			<td>Sutter Instrument</td>
			<td>B100-75-10-PT</td>
			<td>BOROSILICATE GLASS; O.D: 1.0mm, I.D: 0.75mm , length: 10cm, 225Pcs</td>
		</tr>
		<tr>
			<td>Capillary holder</td>
			<td>Eppendorf</td>
			<td>5196 081.005</td>
			<td>Capillary holder 4; for micromanipulation</td>
		</tr>
		<tr>
			<td>Chromic acid mixture</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>037-05415</td>
			<td>For needle washing</td>
		</tr>
		<tr>
			<td>CPA solution</td>
			<td></td>
			<td></td>
			<td>1x EBR containing 20% ethylene glycol and 1M sucrose</td>
		</tr>
		<tr>
			<td>Double-sided tape</td>
			<td>3M</td>
			<td>Scotch w-12</td>
			<td>For glue extracting</td>
		</tr>
		<tr>
			<td>Ephrussi&#8211;Beadle Ringer solution (EBR)</td>
			<td></td>
			<td></td>
			<td>130 mM NaCl, 5 mM KCl, 2 mM CaCl2, and 10 mM Hepes at pH 6.9</td>
		</tr>
		<tr>
			<td>Ethanol (99.5)</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>057-00451</td>
			<td>For embryo collection</td>
		</tr>
		<tr>
			<td>Ethylene glycol</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>054-00983</td>
			<td>For CPA solution</td>
		</tr>
		<tr>
			<td>Falcon 50 mm x 9 mm bacteriological petri dish</td>
			<td>Corning Inc.</td>
			<td>351006</td>
			<td>For embryo collection</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Vigor</td>
			<td>Type5 Titan</td>
			<td>For embryo handling</td>
		</tr>
		<tr>
			<td>Grape juice</td>
			<td>Asahi Soft Drinks Co., LTD.</td>
			<td>Welch&#39;s Grape 100</td>
			<td>For embryo collection</td>
		</tr>
		<tr>
			<td>Grape juice agar plate</td>
			<td></td>
			<td></td>
			<td>50% grape juice, 2% agar, 1% ethanol, 1% acetic acid</td>
		</tr>
		<tr>
			<td>Heptane</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>084-08105</td>
			<td>For glue extracting</td>
		</tr>
		<tr>
			<td>Humidifier</td>
			<td>APIX INTERNATIONAL CO., LTD.</td>
			<td>FSWD2201-WH</td>
			<td>For embryo preparation</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Leica Microsystems GmbH</td>
			<td>Leica&#160;DM IL LED</td>
			<td>For micromanipulation</td>
		</tr>
		<tr>
			<td>Luer-lock glass syringe</td>
			<td>Tokyo Garasu Kikai Co., Ltd.</td>
			<td>0550 14 71 08</td>
			<td>Coat a plunger with silicon oil (FL-100-450CS);for micromanipulation</td>
		</tr>
		<tr>
			<td>Mechanical micromanipulator</td>
			<td>Leica Microsystems GmbH</td>
			<td></td>
			<td>For micromanipulation</td>
		</tr>
		<tr>
			<td>Micro slide glass</td>
			<td>Matsunami Glass Ind., Ltd.</td>
			<td>S-2441</td>
			<td>For embryo aligning</td>
		</tr>
		<tr>
			<td>Microgrinder</td>
			<td>NARISHIGE Group</td>
			<td>Custom order</td>
			<td>EG-401-S combined EG-401 and MF2 (with ocular lens MF2-LE15 ); for needle preparation</td>
		</tr>
		<tr>
			<td>Microscope camera</td>
			<td>Leica Microsystems GmbH</td>
			<td>Leica MC170 HD</td>
			<td>For micromanipulation</td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Merck KGaA</td>
			<td>Eppendorf TransferTip (ES)</td>
			<td>For cryopreservation</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Nacalai Tesque, Inc.</td>
			<td>28514-75</td>
			<td>For EBR solution</td>
		</tr>
		<tr>
			<td>Puller</td>
			<td>NARISHIGE Group</td>
			<td>PN-31</td>
			<td>For needle preparation; the heater level is set to 85.0-98.4, the magnet main level to 57.8, and the magnet sub level to 45.0.</td>
		</tr>
		<tr>
			<td>PVC adhesive tape for electric insulation</td>
			<td>Nitto Denko Corporation</td>
			<td>&#160;J2515</td>
			<td>For embryo-pool frame</td>
		</tr>
		<tr>
			<td>Silicon oil</td>
			<td>Shin-Etsu Chemical, Co, Ltd.</td>
			<td>FL-100-450CS</td>
			<td>For embryo handling</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Nacalai Tesque, Inc.</td>
			<td>31320-05</td>
			<td>For EBR solution</td>
		</tr>
		<tr>
			<td>Sodium hypochlorite solution</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>197-02206</td>
			<td>Undiluted and freshly prepared; for embryo breaching</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Nacalai Tesque, Inc.</td>
			<td>30404-45</td>
			<td>For CPA solution</td>
		</tr>
	</tbody>
</table>

primordial germ cell cryopreservation and revival of drosophila strains

Drosophila strains must be maintained by the frequent transfer of adult flies to new vials. This carries a danger of mutational deterioration and phenotypic changes. Development of an alternative method for long-term preservation without such changes is therefore imperative. Despite previous successful attempts, cryopreservation of Drosophila embryos is still not of practical use because of low reproducibility. Here, we describe a protocol for primordial germ cell (PGC) cryopreservation and strain revival via transplantation of cryopreserved PGCs into agametic Drosophila melanogaster (D. melanogaster) host embryos. PGCs are highly permeable to cryoprotective agents (CPAs), and developmental and morphological variation among strains is less problematic than in embryo cryopreservation. In this method, PGCs are collected from approximately 30 donor embryos, loaded into a needle after CPA treatment, and then cryopreserved in liquid nitrogen. To produce donor-derived gametes, the cryopreserved PGCs in a needle are thawed and then deposited into approximately 15&#160;agametic host embryos. A frequency of at least 15% fertile flies was achieved with this protocol, and the number of progeny per fertile couple was always more than enough to revive the original strain (the average progeny number being 77.2 &#177; 7.1), indicating the ability of cryopreserved PGCs to become germline stem cells. The average number of fertile flies per needle was 1.1 &#177; 0.2, and 9 out of 26 needles produced two or more fertile progeny. It was found that 11 needles are enough to produce 6 or more progeny, in which at least one female and one male are likely included. The agametic host makes it possible to revive the strain quickly by simply crossing newly emerged female and male flies. In addition, PGCs have the potential to be used in genetic engineering applications, such as genome editing.

The efficiency of cryopreserved PGC transplantation has been reported by Asaoka et al.13 and is given in Table 2 for transplantation of PGCs cryopreserved for 1 day or longer in liquid nitrogen. The hatching rate was 168/208 transplanted embryos (80.8%), and the embryo-to-adult viability was 87/208 (41.8%). The frequency of fertile flies was 28/87 (32.2%). This frequency did not differ between PGCs cryopreserved for 8 to 30 days and&#160;for those cryopreserved for 31-150 days (20/57 vs.&#160;8/30, G&#39;&#160;= 0.63, p &#62;0.1, d.f. = 1). The average number of progeny&#160;per couple was 77.2 &#177; 7.1 (n = 18, 28-122), indicating the ability of cryopreserved PGCs to become germline stem cells. Of the 26 needles, 10 produced no fertile progeny, 7 needles produced 1 fertile progeny, 7 needles produced 2 fertile progeny, and 2 needles produced 3 or 4 fertile progeny. The average number of fertile flies per needle was 1.1 &#177; 0.2. Based on this data, with 95% confidence, 11 needles are enough to produce 6 or more progeny, in which at least one female and one male are likely included.
In the above experiments, we used embryos expressing ovo-A mRNA in PGCs (nanos&#62;ovo-A, OvoA_OE embryos) as an agametic host. Out of 669 F1 females and 720 F1 males produced from transplanted nanos&#62;ovo-A couples, there was no escaper that was derived from the host PGCs. Several oskar (osk) mutants are also temperature sensitive agametic20,21. Because an osk mutant with high homozygous viability and the agametic phenotype is no longer available, we recreated the osk[8] missense mutant20 by CRISPR/Cas9-assisted genome editing. These flies were completely agametic (0 escapers out of 230 females and 192 males) at 25 &#176;C, but a few escapers emerged at 23 &#176;C (1 of 248 females and 1 of 290 males). nanos&#62;ovo-A are thus recommended as agametic host embryos. Both UASp-ovo-A and nanos-Gal4 stocks13 will be available soon from the KYOTO Drosophila Stock Center.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65985/65985fig01v2.jpg" /> 
Figure 1: Equipment required. (A) A micromanipulator system to collect and transplant cells. i) inverted microscope, ii) mechanical micromanipulator, iii) syringe, iv) capillary holder, v) three-way stopcock, vi) humidifier, and vii) stereo microscope. (B) A syringe. (C) A three-way stopcock and silicone tubes connect a syringe and a capillary holder. (D) A needle and a capillary holder are attached to a micromanipulator. (E) An embryo-collection cup with an embryo-collection plate (6 cm diameter, 7.7 cm high). (F) A stainless-steel mesh strainer. (G) A container used as a moist chamber with a glass slide. To maintain humidity, place wet paper on the bottom and close the lid. (H) A needle holder with a needle for cryopreservation. (I) A storage rack for cryopreservation and a box with needles. <a href="https://www.jove.com/files/ftp_upload/65985/65985fig01largev2.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/65985/65985fig02v2.jpg" /> 
Figure 2: A PGC-collection glass slide and a cryopreservation needle. (A) A primordial germ cell (PGC)-collection glass slide coated with glue. Dechorionated embryos are aligned in two rows and oriented with their anterior to the right (the side to be manipulated) and ventral side up. An embryo-pool frame is affixed, two drops of cryoprotective agents (CPA) solution are deposited, and the pool is filled with silicone oil. (B) A needle should contain as small an amount of yolk and other contaminants as possible. PGCs are sandwiched between two layers of silicone oil when cryopreserved in liquid nitrogen. <a href="https://www.jove.com/files/ftp_upload/65985/65985fig02largev2.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/65985/65985fig03.jpg" /> 
Figure 3: Making the needle. Three-step tip-polishing method to make a needle with an appropriate hole size and a sharp tip. <a href="https://www.jove.com/files/ftp_upload/65985/65985fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65985/65985fig04v2.jpg" /> 
Figure 4: Embryo collection scheme. After two pre-collections, we usually collect three or four times per day. <a href="https://www.jove.com/files/ftp_upload/65985/65985fig04largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65985/65985fig05.jpg" /> 
Figure 5: Host embryo alignment. Alignment of host embryos on a glass slide. <a href="https://www.jove.com/files/ftp_upload/65985/65985fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65985/65985fig06.jpg" /> 
Figure 6: An overview of the PGC cryopreservation method. An overview of all the steps followed to carry out the primordial germ cell (PGC) cryopreservation. <a href="https://www.jove.com/files/ftp_upload/65985/65985fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="3">Room humidity</td>
		</tr>
		<tr>
			<td></td>
			<td>&#60; 30%</td>
			<td>~ 30%</td>
			<td>&#62; 30%</td>
		</tr>
		<tr>
			<td>Align host embryos 
			(~20 min)</td>
			<td>Use a humidifer for 2 - 10 min</td>
			<td>Use a humidifer intermittently for 1 min</td>
			<td>Do not use a humidifer</td>
		</tr>
		<tr>
			<td>Thaw donor PGCs</td>
			<td>Not applicable</td>
			<td>Not applicable</td>
			<td>Not applicable</td>
		</tr>
		<tr>
			<td>Air dry PGCs</td>
			<td>Omit this step</td>
			<td>Omit this step</td>
			<td>5 min</td>
		</tr>
		<tr>
			<td>Apply silicone oil</td>
			<td>Not applicable</td>
			<td>Not applicable</td>
			<td>Not applicable</td>
		</tr>
		<tr>
			<td>Transplant PGCs</td>
			<td>Not applicable</td>
			<td>Not applicable</td>
			<td>Not applicable</td>
		</tr>
		<tr>
			<td colspan="4">All these steps should be finshed in 50 min.</td>
		</tr>
	</tbody>
</table>
Table 1: Drying of embryos during embryo alignment and PGC thawing.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Donor strain</td>
			<td>Cryopreservation period</td>
			<td>Number of transplanted embryos (A)</td>
			<td>Number of hatched larvae (B) 
			(hatchability, B/A)</td>
			<td>Number of eclosed adults (C) 
			(egg-to-adult viability, C/A)</td>
			<td>Number of fertile adults (D) 
			(frequency of fertile flies, D/C)</td>
		</tr>
		<tr>
			<td>M17</td>
			<td>8 - 30 days</td>
			<td>134</td>
			<td>108 
			(80.6%)</td>
			<td>57 
			(42.5%)</td>
			<td>20 
			(35.1%)</td>
		</tr>
		<tr>
			<td>M17</td>
			<td>31 - 150 days</td>
			<td>74</td>
			<td>60 
			(81.1%)</td>
			<td>30 
			(40.5%)</td>
			<td>8 
			(26.7%)</td>
		</tr>
		<tr>
			<td colspan="6">M17: yw; TM6B, P{Dfd-GMR-nvYFP}4, Sb[1] Tb[1] ca[1]/ Pri[1]</td>
		</tr>
	</tbody>
</table>
Table 2: Efficiency of cryopreserved PGC transplantation. This table is modified from13. All data are&#160;from agametic hosts.

Watch this Scientific Journal Video about PGC Cryopreservation â€“ An Alternative Approach Towards Long-Term Preservation of Drosophila Strains at JoVE.com

Author Spotlight: PGC Cryopreservation &#8211; An Alternative Approach Towards Long-Term Preservation of Drosophila Strains 

A long-term preservation method for Drosophila strains as an alternative to the frequent transfer of adult flies to fresh food vials is highly desirable. This protocol describes the cryopreservation of Drosophila primordial germ cells and strain revival via their transplantation to agametic host embryos.

Primordial Germ Cell Cryopreservation and Revival of Drosophila Strains

Drosophila strains must be maintained by the frequent transfer of adult flies to new vials. This carries a danger of mutational deterioration and ...

primordial-germ-cell-cryopreservation-revival-drosophila

Research

JoVE Journal

Genetics

2.2K Views.  Kyoto Institute of Technology. A long-term preservation method for Drosophila strains as an alternative to the frequent transfer of adult flies to fresh food vials is highly desirable. This protocol describes the cryopreservation of Drosophila primordial germ cells and strain revival via their transplantation to agametic host embryos.

Video: Author Spotlight: PGC Cryopreservation – An Alternative Approach Towards Long-Term Preservation of Drosophila Strains 

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

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 ...

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.

Recent advances in the ability to genetically manipulate somatic cell lines hold great potential for basic and applied research. Here, we present two approaches for CRISPR/Cas9 generated knockout production and screening in mammalian cell lines, with and without the use of selectable markers.

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in humans, but more tools and research efforts are needed to better elucidate the mechanisms involved. For over a century, the genetically highly tractable model of Drosophila has been instrumental in the discovery of key genes and molecular pathways that proved to be highly conserved across species. Many biological processes and disease mechanisms are functionally conserved in the fly, such as development (e.g., body plan, heart), cancer, and neurodegenerative disease. Recently, the study of obesity and secondary pathologies, such as heart disease in model organisms, has played a highly critical role in the identification of key regulators involved in metabolic syndrome in humans.
Here, we propose to use this model organism as an efficient tool to induce obesity, i.e., excessive fat accumulation, and develop an efficient protocol to monitor fat content in the form of TAGs accumulation. In addition to the highly conserved, but less complex genome, the fly also has a short lifespan for rapid experimentation, combined with cost-effectiveness. This paper provides a detailed protocol for High Fat Diet (HFD) feeding in Drosophila to induce obesity and a high throughput triacylglyceride (TAG) assay for measuring the associated increase in fat content, with the aim to be highly reproducible and efficient for large-scale genetic or chemical screening. These protocols offer new opportunities to efficiently investigate regulatory mechanisms involved in obesity, as well as provide a standardized platform for drug discovery research for rapid testing of the effect of drug candidates on the development or prevention of obesity, diabetes and related metabolic diseases.

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

This is a high fat diet feeding protocol to induce obesity in Drosophila, a model for understanding fundamental molecular mechanisms implicated in lipotoxicity. It also provides a high throughput triacylglyceride assay for measuring fat accumulation in Drosophila and potentially other (insect) models under various dietary, environmental, genetic or physiological conditions.

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in ...

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the African clawed frog, Xenopus, a longstanding model organism for biomedical research. Traditional breeding schemes for creating homozygous mutant lines with CRISPR/Cas9-targeted mutagenesis have several time-consuming and laborious steps. To facilitate the creation of mutant embryos, particularly to overcome the obstacles associated with knocking out genes that are essential for embryogenesis, a new method called leapfrogging was developed. This technique leverages the robustness of Xenopus embryos to &#34;cut and paste&#34; embryological methods. Leapfrogging utilizes the transfer of primordial germ cells (PGCs) from efficiently-mutagenized donor embryos into PGC-ablated wildtype siblings. This method allows for the efficient mutation of essential genes by creating chimeric animals with wildtype somatic cells that carry a mutant germline. When two F0 animals carrying &#34;leapfrog transplants&#34; (i.e., mutant germ cells) are intercrossed, they produce homozygous, or compound heterozygous, null F1 embryos, thus saving a full generation time to obtain phenotypic data. Leapfrogging also provides a new approach for analyzing maternal effect genes, which are refractory to F0 phenotypic analysis following CRISPR/Cas9 mutagenesis. This manuscript details the method of leapfrogging, with special emphasis on how to successfully perform PGC transplantation.

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

Genes essential for survival pose technical hurdles for creating mutant lines. Leapfrogging circumvents lethality by combining genome editing with primordial germ cell transplantation to create wild-type animals carrying germline mutations. Leapfrogging also permits the efficient generation of homozygous null mutants in the F1 generation. Here, the transplantation step is demonstrated.

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the ...

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene&#160;NDC80&#160;during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in&#160;other yeast strains and growth conditions.

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is inherently a single-cell assay, however prior to molecular analysis, the target cells are often prospectively isolated in bulk, thereby losing single-cell resolution. Single-cell gene expression analysis provides a means to understand molecular differences between individual cells in heterogeneous cell populations. In bulk cell analysis an overrepresentation of a distinct cell type results in biases and occlusions of signals from rare cells with biological importance. By utilizing FACS index sorting coupled to single-cell gene expression analysis, populations can be investigated without the loss of single-cell resolution while cells with intermediate cell surface marker expression are also captured, enabling evaluation of the relevance of continuous surface marker expression. Here, we describe an approach that combines single-cell reverse transcription quantitative PCR (RT-qPCR) and FACS index sorting to simultaneously characterize the molecular and immunophenotypic heterogeneity within cell populations.
In contrast to single-cell RNA sequencing methods, the use of qPCR with specific target amplification allows for robust measurements of low-abundance transcripts with fewer dropouts, while it is not confounded by issues related to cell-to-cell variations in read depth. Moreover, by directly index-sorting single-cells into lysis buffer this method, allows for cDNA synthesis and specific target pre-amplification to be performed in one step as well as for correlation of subsequently derived molecular signatures with cell surface marker expression. The described approach has been developed to investigate hematopoietic single-cells, but have also been used successfully on other cell types.
In conclusion, the approach described herein allows for sensitive measurement of mRNA expression for a panel of pre-selected genes with the possibility to develop protocols for subsequent prospective isolation of molecularly distinct subpopulations.

Bulk gene expression measurements cloud individual cell differences in heterogeneous cell populations. Here, we describe a protocol for how single-cell gene expression analysis and index sorting by Florescence Activated Cell Sorting (FACS) can be combined to delineate heterogeneity and immunophenotypically characterize molecularly distinct cell populations.

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is ...

Preparation and Gene Modification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, underlies the only known cure of human immunodeficiency virus (HIV-1) infection, and shows immense promise in the treatment of genetic diseases such as beta thalassemia. Our group has developed a protocol to model HSPC gene therapy in nonhuman primates (NHPs), allowing scientists to optimize many of the same reagents and techniques that are applied in the clinic. Here, we describe methods for purifying CD34+ HSPCs and long-term persisting hematopoietic stem cell (HSC) subsets from primed bone marrow (BM). Identical techniques can be employed for the purification of other HSPC sources (e.g., mobilized peripheral blood stem cells [PBSCs]). Outlined is a 2 day protocol in which cells are purified, cultured, modified with lentivirus (LV), and prepared for infusion back into the autologous host. Key readouts of success include the purity of the CD34+ HSPC population, the ability of purified HSPCs to form morphologically distinct colonies in semisolid media, and, most importantly, gene modification efficiency. The key advantage to HSPC gene therapy is the ability to provide a source of long-lived cells that give rise to all hematopoietic cell types. As such, these methods have been used to model therapies for cancer, genetic diseases, and infectious diseases. In each case, therapeutic efficacy is established by enhancing the function of distinct HSPC progeny, including red blood cells, T cells, B cells, and/or myeloid subsets. The methods to isolate, modify, and prepare HSPC products are directly applicable and translatable to multiple diseases in human patients.

The goal of this protocol is to isolate nonhuman primate CD34+ cells from primed bone marrow, to gene-modify these cells with lentiviral vectors, and to prepare a product for infusion into the autologous host. The total protocol length is approximately 48 h.

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, ...

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media.

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

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 ...