cryopreservation of primordial germ cells collected from drosophila donor embryos

用于果 蝇 菌株长期保存的 PGC 冷冻保存

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.

作者聚焦：PGC 冷冻保存 – 一种长期保存果蝇菌株的替代方法 

果蝇菌株的原始生殖细胞冷冻保存和复兴

Drosophila strain must be maintained by the frequent transfer of adult flies to new vials. This creates a danger of mutational deterioration and genetic changes. We aim to develop an alternative method for the long-term preservation of Drosophila strains without such changes.Compare with in vivo editing, genome editing of primordial germ cells, or PGCs, could increase knock-in inefficiency and make more complicated genomic changes. Thus, a wide application of PGC manipulation facilitates genetic engineering in many fields. We now focus on xenotransplantation.By using Drosophila melanogaster as a surrogate host, we have succeeded in reviving stock of cell species in the melanogaster species subgroup. By addressing xenotransplantation barriers and finding possible solutions, we would like to explore the potential application of PGC transplantation and cryopreservation.

Watch this Scientific Journal Video about Cryopreservation of Primordial Germ Cells Collected from Drosophila Donor Embryos at JoVE.com

Cryopreservation of Primordial Germ Cells Collected from Drosophila Donor Embryos  

从 果蝇 供体胚胎中收集的原始生殖细胞的冷冻保存

继续进行原始生殖细胞或PGC的冷冻保存。将供体胚胎的PGC收集并沉积在胚胎表面后。用冷冻保护剂或 CPA 清洗移植针头，然后将新鲜的 CPA 装入针头中。将加载的 CPA 添加到沉积在胚胎上的 PGC 中，同时确保 CPA 的体积与 PGC 的体积相同。 添加 CPA 后一到两秒，从 PGC 簇中去除多余的 CPA。接下来，清空针头并用硅油装入五秒钟或更长时间。将所有收集的 PGC 加载到针中，然后再次加载硅油 5 秒钟或更长时间。确保 PGC 夹在两层硅油之间。使用软薄纸将针头从显微操纵器上拆下，吸干针头上的油，不要将针尖与纸巾接触。将针头连接到针架上，并使用乙烯基胶带将其锁定在底部的位置。将针头朝下，将其浸入液氮中，快速冷冻支架。在液体停止从架子中嘶嘶作响之前，不要松开支架。将支架存放在液相区域的液氮储罐中。

cryopreservation-primordial-germ-cells-collected-from-drosophila

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JoVE Journal

Genetics

Cryopreservation of Primordial Germ Cells Collected from Drosophila Donor Embryos

268 次观看.  Kyoto Institute of Technology. 继续进行原始生殖细胞或PGC的冷冻保存。将供体胚胎的PGC收集并沉积在胚胎表面后。用冷冻保护剂或 CPA 清洗移植针头，然后将新鲜的 CPA 装入针头中。将加载的 CPA 添加到沉积在胚胎上的 PGC 中，同时确保 CPA 的体积与 PGC 的体积相同。 添加 CPA 后一到两秒，从 PGC 簇中去除多余的 CPA。接下来，清空针头并用硅油装入五秒钟或更长时间。将所有收集的 PGC 加载到针中?...

视频: Cryopreservation of Primordial Germ Cells Collected from Drosophila Donor 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 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.

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 ...

科研

遗传学

Collection of Primordial Germ Cells from Drosophila Donor Embryos

Collection of Primordial Germ Cells from Drosophila Donor Embryos  

Proceed to collect the primordial germ cells or PGCs from the dross filia embryos. After assembling the micro manipulator system. While working under a stereo microscope, use forceps to transfer the embryos.Align the coated embryos in two rows on a PGC collection glass slide along two reference lines. Orient the embryos with their anterior to the right and ventral side up. Gently move the transplantation needle tip to the anterior end of an embryo.Penetrate the embryo by moving the microscope stage and retract the needle slightly. When the tip reaches the posterior end. Discharge any yoke in the needle inside the somatic cell layer.Move the needle tip to the posterior pole and gently load the PGCs into the needle without taking much time. Pull the needle out of the embryo quickly. Discharge yolk and other contaminants from the needle while keeping the PGCs.Load clean silicone oil from the pool into the needle. Before collecting PGCs from a new embryo, deposit as much silicone oil as possible inside the somatic cell layer while keeping the loaded PGCs in the needle. After collecting PGCs from all embryos in a row, deposit the PGCs onto the surface of an embryo and unload any yoke or other contaminants onto another neighboring embryo.In a similar manner, collect and combine PGCs from the embryos in the second row.

从 果蝇 供体胚胎中收集原始生殖细胞

Proceed for the cryo-preservation of the primordial germ cells or PGCs. After collecting and depositing the PGCs from the donor embryos on the surface of an embryo. Wash the transplantation needle with a cryo protective agent or CPA and load fresh CPA into the needle.Add the loaded CPA to the PGCs deposited on the embryo while ensuring the volume of CPA is equivalent to that of the PGCs. Remove excess CPA from the cluster of PGCs one to two seconds after CPA addition. Next, empty the needle and load it with silicone oil for five seconds or longer.Load all the collected PGCs into the needle, followed by loading silicone oil again for five seconds or longer. Ensure the PGCs are sandwiched between two layers of silicone oil. Detach the needle from the micro manipulator using soft tissue paper, blot the oil off the needle surface without touching the needle tip with the tissue.Attach the needle to a needle holder and using vinyl tape, lock it in position at the base. 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.Store the holder in a liquid nitrogen storage tank in the liquid phase area.

Thawing and Transplantation of Cryopreserved Primordial Germ Cells for Drosophila Strain Revival

Thawing and Transplantation of Cryopreserved Primordial Germ Cells for Drosophila Strain Revival  

To begin, align the stage five egg mimetic Drosophila host embryos on a transplantation glass slide. While orienting their posterior to the right and ventral to the top, line up approximately 30 embryos in two rows in 20 minutes. Thaw the cryopreserved PGCs in the needle by submerging the holder with the needle pointing downward into EBR solution at room temperature.Keep it submerged for 10 seconds. Place the transplantation glass slide on the microscope stage. Attach the freeze thawed needle to the capillary holder and bring the first embryo into the left row and the needle tip into the same focal plane.Using a 20x objective lens, position the needle tip gently at the surface of the posterior end of the embryo. Prod the outside of each embryo gently, and confirm that they slowly return to their original shape. Penetrate an embryo from the posterior pole using the needle.Gently deposit approximately 10 to 20 PGCs inside the posterior pole, specifically between the vitelline membrane and the somatic cell layer, and retract the needle from the embryo before repeating the same for subsequent embryos. Finally, to revive the strain, cross newly emerged females and males.