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.
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 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 ± 7.1), indicating the ability of cryopreserved PGCs to become germline stem cells. The average number of fertile flies per needle was 1.1 ± 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 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.
1. Equipment preparation
2. Collection and cryopreservation of PGCs
3. Thawing and transplanting PGCs
4. Incubating embryos and restoring donor strains
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 for those cryopreserved for 31-150 days (20/57 vs. 8/30, G' = 0.63, p >0.1, d.f. = 1). The average number of progeny per couple was 77.2 ± 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 ± 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>ovo-A, OvoA_OE embryos) as an agametic host. Out of 669 F1 females and 720 F1 males produced from transplanted nanos>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 °C, but a few escapers emerged at 23 °C (1 of 248 females and 1 of 290 males). nanos>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.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Figure 3: Making the needle. Three-step tip-polishing method to make a needle with an appropriate hole size and a sharp tip. Please click here to view a larger version of this figure.
Figure 4: Embryo collection scheme. After two pre-collections, we usually collect three or four times per day. Please click here to view a larger version of this figure.
Figure 5: Host embryo alignment. Alignment of host embryos on a glass slide. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Room humidity | |||
< 30% | ~ 30% | > 30% | |
Align host embryos (~20 min) |
Use a humidifer for 2 – 10 min | Use a humidifer intermittently for 1 min | Do not use a humidifer |
Thaw donor PGCs | Not applicable | Not applicable | Not applicable |
Air dry PGCs | Omit this step | Omit this step | 5 min |
Apply silicone oil | Not applicable | Not applicable | Not applicable |
Transplant PGCs | Not applicable | Not applicable | Not applicable |
All these steps should be finshed in 50 min. |
Table 1: Drying of embryos during embryo alignment and PGC thawing.
Donor strain | Cryopreservation period | Number of transplanted embryos (A) | Number of hatched larvae (B) (hatchability, B/A) |
Number of eclosed adults (C) (egg-to-adult viability, C/A) |
Number of fertile adults (D) (frequency of fertile flies, D/C) |
M17 | 8 – 30 days | 134 | 108 (80.6%) |
57 (42.5%) |
20 (35.1%) |
M17 | 31 – 150 days | 74 | 60 (81.1%) |
30 (40.5%) |
8 (26.7%) |
M17: yw; TM6B, P{Dfd-GMR-nvYFP}4, Sb[1] Tb[1] ca[1]/ Pri[1] |
Table 2: Efficiency of cryopreserved PGC transplantation. This table is modified from13. All data are from agametic hosts.
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 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 authors have nothing to disclose.
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 to S.K.
Acetic acid | FUJIFILM Wako Pure Chemical Corporation | 017-00256 | For embryo collection |
Agar powder | FUJIFILM Wako Pure Chemical Corporation | 010-08725 | For embryo collection |
Calcium chloride | FUJIFILM Wako Pure Chemical Corporation | 038-24985 | For EBR solution |
Capillary | Sutter Instrument | B100-75-10-PT | BOROSILICATE GLASS; O.D: 1.0mm, I.D: 0.75mm , length: 10cm, 225Pcs |
Capillary holder | Eppendorf | 5196 081.005 | Capillary holder 4; for micromanipulation |
Chromic acid mixture | FUJIFILM Wako Pure Chemical Corporation | 037-05415 | For needle washing |
CPA solution | 1x EBR containing 20% ethylene glycol and 1M sucrose | ||
Double-sided tape | 3M | Scotch w-12 | For glue extracting |
Ephrussi–Beadle Ringer solution (EBR) | 130 mM NaCl, 5 mM KCl, 2 mM CaCl2, and 10 mM Hepes at pH 6.9 | ||
Ethanol (99.5) | FUJIFILM Wako Pure Chemical Corporation | 057-00451 | For embryo collection |
Ethylene glycol | FUJIFILM Wako Pure Chemical Corporation | 054-00983 | For CPA solution |
Falcon 50 mm x 9 mm bacteriological petri dish | Corning Inc. | 351006 | For embryo collection |
Forceps | Vigor | Type5 Titan | For embryo handling |
Grape juice | Asahi Soft Drinks Co., LTD. | Welch's Grape 100 | For embryo collection |
Grape juice agar plate | 50% grape juice, 2% agar, 1% ethanol, 1% acetic acid | ||
Heptane | FUJIFILM Wako Pure Chemical Corporation | 084-08105 | For glue extracting |
Humidifier | APIX INTERNATIONAL CO., LTD. | FSWD2201-WH | For embryo preparation |
Inverted microscope | Leica Microsystems GmbH | Leica DM IL LED | For micromanipulation |
Luer-lock glass syringe | Tokyo Garasu Kikai Co., Ltd. | 0550 14 71 08 | Coat a plunger with silicon oil (FL-100-450CS);for micromanipulation |
Mechanical micromanipulator | Leica Microsystems GmbH | For micromanipulation | |
Micro slide glass | Matsunami Glass Ind., Ltd. | S-2441 | For embryo aligning |
Microgrinder | NARISHIGE Group | Custom order | EG-401-S combined EG-401 and MF2 (with ocular lens MF2-LE15 ); for needle preparation |
Microscope camera | Leica Microsystems GmbH | Leica MC170 HD | For micromanipulation |
Needle holder | Merck KGaA | Eppendorf TransferTip (ES) | For cryopreservation |
Potassium chloride | Nacalai Tesque, Inc. | 28514-75 | For EBR solution |
Puller | NARISHIGE Group | PN-31 | 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. |
PVC adhesive tape for electric insulation | Nitto Denko Corporation | J2515 | For embryo-pool frame |
Silicon oil | Shin-Etsu Chemical, Co, Ltd. | FL-100-450CS | For embryo handling |
Sodium chloride | Nacalai Tesque, Inc. | 31320-05 | For EBR solution |
Sodium hypochlorite solution | FUJIFILM Wako Pure Chemical Corporation | 197-02206 | Undiluted and freshly prepared; for embryo breaching |
Sucrose | Nacalai Tesque, Inc. | 30404-45 | For CPA solution |