Production Of Germline Chimeras Research Articles

First study on repeatable culture of primordial germ cells from various embryonic regions with giant feeder cells in Japanese quail (Coturnix japonica)

Japanese quail (JQ, Coturnix japonica) is a farmed animal with a high economic value and has been used extensively as an avian model for research. Germline chimera production based on cryopreserved primordial germ cells (PGCs) is possible for conservation management of quail breeds as successful isolation has been reported of PGCs from their blood and gonads. However, the repeatable cultivation protocol has not been elucidated yet, which has hindered technological development. The current study characterized cultivation of pregonadal PGCs isolated from embryonic parts; embryonic blood (cPGCs), whole embryonic tissues (tPGCs), parts of tail buds (tbPGCs), and a mixture of blood and tail bud tissues (ctbPGCs). The results showed that the cultivation system required the presence of specific embryonic cells to act as a feeder for JQ-PGCs and that such a system facilitated more successful cultivation, as shown by the percentages of isolation and cultivation in tbPGCs (100%, 100%, respectively), tPGCs (60%, 55%, respectively), and ctbPGCs (60%, 30%, respectively), but not in cPGCs (0%) cultured on a mitomycin-treated JQ feeder cell-line. Once the co-culture system had been established, the PGCs could be propagated for at least 5 months. These PGCs expressed germ cell-specific markers (DAZL and CVH) and could colonize embryonic gonads. Conclusively, the isolation of pregonadal PGCs and their long-term cultivation in vitro requires a unique embryonic cell, giant cell feeder, that is indispensable for the proliferation of PGCs. Characterization of cell signaling sustaining a mutual interaction between the PGCs and the specific feeder cells will elucidate a superior environment for in vitro cultivation, as well as support the minimal transfer of used xenobiotics in chimera production.

Current Approaches to and the Application of Intracytoplasmic Sperm Injection (ICSI) for Avian Genome Editing.

Poultry are one of the most valuable resources for human society. They are also recognized as a powerful experimental animal for basic research on embryogenesis. Demands for the supply of low-allergen eggs and bioreactors have increased with the development of programmable genome editing technology. The CRISPR/Cas9 system has recently been used to produce transgenic animals and various animals in the agricultural industry and has also been successfully adopted for the modification of chicken and quail genomes. In this review, we describe the successful establishment of genome-edited lines combined with germline chimera production systems mediated by primordial germ cells and by viral infection in poultry. The avian intracytoplasmic sperm injection (ICSI) system that we previously established and recent advances in ICSI for genome editing are also summarized.

Gonadal Germ Cell Migration and Proliferation after Transfer in Developing Chicken Embryos.

A germline chimera is a useful model for developing and differentiating germ cells in vivo. Gonadal germ cells (GGCs) collected from chicken embryonic gonads may be used to produce germline chimeras as donor cells. However, the migratory and proliferative abilities of GGCs after transfer into recipient embryos are unclear. Here, the migratory and proliferative abilities of GGCs collected from 7-day-old White Leghorn embryos and fluorescently labeled were analyzed following transfer into the dorsal aorta of 2.5-day-old Rhode Island Red (RIR) embryos. Five days after transfer, the numbers of male and female GGCs were significantly higher in the RIR gonads than those in non-gonadal RIR organs when 50 GGCs were transferred per embryo. To analyze the temporal migration of GGCs in intermediate mesoderm, 50 GGCs were again transferred. The numbers of male and female GGCs in RIR gonads increased significantly from days 3 to 6 after transfer. To analyze GGC migration and proliferation in the gonads, a single GGC was transferred into 100 male and 100 female embryos. Five days after transfer, the frequencies of settled and proliferated GGCs were 37% (37/100) and 24% (24/100) in males, and 23% (23/100) and 8% (8/100) in females, respectively. Thus, GGCs are a heterogeneous cell population that may or may not have migratory and proliferative abilities. The heterogeneity of GGCs may be greater in females than that in males. When 50 GGCs were transplanted, almost all those present in embryos had settled and proliferated in the gonads and mesonephros. The migratory and proliferative abilities of GGCs in recipient gonads were considerably diverse in individual GGCs or between donor sexes.

Transplantation and enrichment of busulfan-resistant primordial germ cells into adult testes for efficient production of germline chimeras in songbirds†.

Zebra finch is a unique model for behavioral, neural, and genomic studies of vocal learning. Several transgenic zebra finches have been produced, although the germline transmission efficiencies are reportedly low. Recently, there have been attempts to produce germline chimeras using primordial germ cells (PGCs). However, this has been hampered by difficulties associated with the manipulation of the small eggs and the fact that the zebra finch is an altricial species that requires parental care after birth, unlike precocial chickens. Consequently, it is difficult to transplant PGCs into embryos and maintain the chimeras. Here, we developed a busulfan-mediated system for transplantation of PGCs into adult testes, to produce germline chimeras with an improved germline transmission capacity. We established microsomal glutathione-S-transferase II (MGSTII)-overexpressing PGCs that are resistant to busulfan, which induces germ cell-specific cytotoxicity, and transplanted them into testes rendered temporarily infertile by busulfan. The recipients were given a second dose of busulfan to deplete endogenous germ cells and enrich the transplanted cells, and donor cell-derived spermatogenesis was accomplished. This method requires fewer recipients due to higher survival rates, and there is no need to wait for maturation of the founders, which is required when transplanting PGCs into embryos. These results are expected to improve transgenic zebra finch production.

Primordial germ cell identification and traceability during the initial development of the Siluriformes fish Pseudopimelodus mangurus.

Primordial germ cells (PGCs) are responsible for generating all germ cells. Therefore, they are essential targets to be used as a tool for the production of germline chimeras. The labeling and route of PGCs were evaluated during the initial embryonic development of Pseudopimelodus mangurus, using whole-mount in situ hybridization (WISH) and mRNA microinjection in zygotes. A specific antisense RNA probe constituted by a partial coding region from P. mangurus nanos3 mRNA was synthesized for the WISH method. RNA microinjection was performed using the GFP gene reporter regulated by translation regulatory P. mangurus buc and nanos3 3'UTR sequences, germline-specific markers used to describe in vivo migration of PGCs. Nanos3 and buc gene expression was evaluated in tissues for male and female adults and initial development phases and larvae from the first to seventh days post-hatching. The results from the WISH technique indicated the origin of PGCs in P. mangurus from the aggregations of nanos3 mRNA in the cleavage grooves and the signals obtained from nanos3 probes corresponded topographically to the migratory patterns of the PGCs reported for other fish species. Diffuse signals were observed in all blastomeres until the 16-cell stage, which could be related to the two sequences of the nanos3 3'UTR observed in the P. mangurus unfertilized egg transcriptome. Microinjection was not successful using GFP-Dr-nanos1 3'UTR mRNA and GFP-Pm-buc 3'UTR mRNA and allowed the identification of potential PGCs with less than 2% efficiency only and after hatching using GFP-Pm-nanos3 3'UTR. Nanos3 and buc gene expression was reported in the female gonads and from fertilized eggs until the blastula phase. These results provide information about the PGC migration of P. mangurus and the possible use of PGCs for the future generation of germline chimeras to be applied in the conservation efforts of Neotropical Siluriformes species. This study can contribute to establishing genetic banks, manipulating organisms, and assisting in biotechnologies such as transplanting germ cells in fish.

Current Approaches and Applications in Avian Genome Editing.

Advances in genome-editing technologies and sequencing of animal genomes enable researchers to generate genome-edited (GE) livestock as valuable animal models that benefit biological researches and biomedical and agricultural industries. As birds are an important species in biology and agriculture, their genome editing has gained significant interest and is mainly performed by using a primordial germ cell (PGC)-mediated method because pronuclear injection is not practical in the avian species. In this method, PGCs can be isolated, cultured, genetically edited in vitro, and injected into a recipient embryo to produce GE offspring. Recently, a couple of GE quail have been generated by using the newly developed adenovirus-mediated method. Without technically required in vitro procedures of the PGC-mediated method, direct injection of adenovirus into the avian blastoderm in the freshly laid eggs resulted in the production of germ-line chimera and GE offspring. As more approaches are available in avian genome editing, avian research in various fields will progress rapidly. In this review, we describe the development of avian genome editing and scientific and industrial applications of GE avian species.

Production of Germ-Line Chimeras in Zebrafish.

The induction of germ-line chimerism in fish is a strategy for the reproduction of endangered or genetically valuable fish species. Chimeras can be created by transplanting a single primordial germ cell or multiple blastomeres from a donor into a sterile host embryo. When the host reaches sexual maturity, it will produce donor-originating gametes throughout its reproductive life span. This technique provides unique experimental conditions for basic biology research in model fish species like zebrafish. The success of cell transplantation relies on the effective sterilization of host embryos, the correct identification of developing germ cells, and the synchronization of migratory cues between the host and the transplanted cells. Developments in non-transgenic methods of germ cell ablation and identification have made germ cell transplantation more applicable to use in conservation and aquaculture. In this chapter, we provide a protocol for germ cell labeling by injection of chimeric RNA or FITC-dextran, the sterilization of host embryos using an antisense morpholino oligonucleotide, and two methods for producing germ-line chimeras in zebrafish: single primordial germ cell transplant and blastomere transplant.

Germ Cell Transplantation in Avian Species.

Germcell transplantation technology has played a critical role in germline modification and preservation of genetic resources. Several germcell transplantation systems have been developed, including sperm, oocyte, or germline stemcell transplantation systems in mammals. Meanwhile, in avian species, this has mostly relied on primordial germcell (PGC) transplantation for efficient germline transmission. In this chapter, we describe how to isolate PGCs from avian embryos and produce germline chimeras through transplantation of donor PGCs to recipient embryos.

Production of Transgenic Chickens Using Cultured Primordial Germ Cells and Gonocytes.

The unique characteristics of the avian embryo, with its large opaque yolk, have necessitated the development of different approaches to transgenesis from those that have been successful in mammalian species. Genetic modification of birds was greatly advanced by the ability to grow long-term cultures of primordial germ cells (PGCs). These cells are obtained from embryos, established in culture, and can be propagated without losing the ability to contribute to the germline when reintroduced into a host animal. PGCs can be genetically modified in culture using traditional transfection and selection techniques, including gene targeting and site-specific nuclease approaches. Here, we describe our methods for deriving cell lines, long-term culture, genetic modification, production of germline chimeras and obtaining fully transgenic birds with the desired genetic modifications.

Effectiveness of DMSO concentration on recovery rate and viability of primordial germ cell of Gaok chicken

<p class="abstrak2">Recent technological developments to produce germ line chimeras with primordial germ cell (PGC) transfer into the recipient embryo provide an opportunity to conserve and retrieval of chicken genetic resources in complete form. The study was conducted to obtain the most effective DMSO percentage to recovery rate and viability of Gaok chicken PGC after freezing which will later be feasible to be transferred. In this study, the eggs of Gaok chicken were incubated for about 2.5 - 3 days to obtain embryos at stages 14 - 16. Blood retrieval was done through the dorsal aorta using micropipettes under microscope. The procedure of PGC isolation of Gaok chicken with centrifugation gradient was using nycodenz as a substance. Commercially available cryoprotectants (dimethyl sulfoxide = DMSO) were used for PGC freezing. Isolated and frozen PGCs of Gaok chicken were diluted with cryoprotectants containing 2.5; 5 and 10% DMSO in fetal bovine serum (FBS). The recovery rate of 2.5; 5 and 10% DMSO concentration were 36.4; 48.2 and 48 % respectively. The viability of PGC after freezing was significantly higher for 5% DMSO compared with 2.5% DMSO (P<0.05), but not different from 10% DMSO. It can be concluded that the concentration DMSO of 5 % was effectve contration in freezing Gaok chicken PGC.</p>

Manipulating the Avian Egg: Applications for Embryo Transfer, Transgenics, and Cloning

In vitro production of germline chimeras and avian cloning may utilise the transfer of avian embryos from their original eggshell to a surrogate eggshell for culture during incubation. Such embryo transfer is valuable for avian cloning as the only alternative would be to transfer the cloned avian embryos into the infundibulum of recipient birds. Given the advances in paleogenomics, synthetic biology, and gene editing, a similar approach might be used to generate extinct species, i.e. de-extinction. One objective of the present research was to examine if ratite eggs could be manipulated via windowing and sham injection, similar to that which could allow for avian genome manipulation and subsequent development. The efficiency of interspecific avian embryo transfer using Chicken ( Gallus gallus domesticus) donor eggs and Turkey ( Meleagris gallopavo) recipient eggshells was also investigated. Egg windowing and embryo transfer techniques utilised in the present research were adapted from those found in the scientific literature. Presumed fertile eggs from Rhode Island Red ( n = 40), Silkie ( n = 2), and White Leghorn Chickens ( n = 18), Turkey ( n = 48), Emu ( Dromaius novaehollandiae) ( n = 79), and Ostrich ( Struthio camelus) ( n = 89) were used in this research. Of the 41 Chicken eggs used for transfers into recipient Turkey eggshells, only one (2.4%) produced a chick. Of 31 windowed Emu eggs, one embryo survived for 25 d but no chicks were produced. Of 36 windowed Ostrich eggs, one embryo survived and hatched. The efficiency of the windowing and embryo transfers to produce chicks was low and further refinements are needed. Importantly, the results herein establish that manipulating ratite embryos is possible.

Isolation and Number of Gonadal Primordial Germ Cells (Gonadal PGCs) on the Stages of Early Embryonic Development of KUB Chicken

Primordial germ cells (PGCs) are cells that will differentiate themselves into spermatogonia in the testis or oogonia in the ovary. Primordial germ cells arise from epiblast and circulate through the bloodstream and finally entering gonadal anlage. The aim of this study was to determine the number of gonadal PGCs of KUB chicken at different development stages. Sixty KUB chicken fertile eggs were divided into four groups (6, 7, 8, and 9 days incubation periods), and incubated at 38 oC with a humidity of 60%. Harvesting was synchronized to the embryonic development at 6-9 d. Gonads were collected using sharp tweezers, and were placed in Eppendorf tube 1.5 mL containing 500 µL PBS [-]. Gonadal PGCs were purified using PBS [-]. The results showed that the average number of gonadal PGCs at 6, 7, 8, and 9 d were 113.7; 143.5; 92.9; and 85.7 cells per embryo, respectively. Number of gonadal PGCs per embryo of KUB chicken were significantly affected by stage of embryonic development (P<0.05), which reached a peak at day 7 of incubation, so that the isolation and collection of PGCs from the gonads were recommended at day 7 of incubation. This information is useful in production of germline chimera of other Indonesian local chickens.

Migration of primordial germ cells during late embryogenesis of pikeperch Sander lucioperca relative to blastomere transplantation

Pikeperch Sander lucioperca is a valuable fish in Europe, and basic information about its embryonic development, especially primordial germ cell (PGC) migration, is important for use in biotechnology. We categorized pikeperch embryonic development into six stages as in other fish species: zygote, cleavage, blastula, gastrula, segmentation, and hatching and described PGC migration. PGCs were visualized by injection of synthesized green fluorescent protein (GFP) within the 3’untranslated region (UTR) mRNA of nanos3. GFP-positive PGCs appeared in all embryos at approximately 100% epiboly. Time-lapse imaging revealed the PGC migration pattern from their initial appearance to location at the gonadal ridge. We conducted blastomere transplantation (BT) at the blastula stage. Donor embryos were labelled with GFP-nos3 3’UTR mRNA and tetramethylrhodamine dextran to label PGCs and somatic cells, respectively. Twelve BT chimeras were produced, with eight surviving to hatching. All exhibited donor-derived somatic cells in the developing body. The PGCs from donor embryos were observed to migrate towards the gonad region of the host embryos. Our results indicated that BT can be successfully applied in pikeperch, and these findings may be useful to produce germline chimeras in percids.

Isolation, Characterization, and In Vitro Culturing of Spermatogonial Stem Cells in Japanese Quail (Coturnix japonica).

We reported previously testis-mediated germline chimera production and characterization of germline stem cell-like cells from chicken testes. The present study aimed to establish an in vitro system for culture of quail spermatogonial stem cells (SSCs) for practical applications in germline preservation and transgenesis. Testicular cells (TCs) from juvenile (4 weeks old) or adult (8 weeks old) quail testis were isolated using sequential enzymatic digestion. The percentages of viability of isolated TCs were 91.00% ± 2.12% and 88.00% ± 1.87% in juvenile and adult testes, respectively, and immunohistochemical evaluation indicated the expression of integrin alpha-6 (ITGA6), GDNF family receptor alpha-1 (GFRA1), and Deleted in azoospermia-like (DAZL) in specific TCs. SSCs were purified by differential plating of TCs and then subjected to quantitative reverse transcription-polymerase chain reaction, which showed differential expression of SSC-specific, and germness and stemness-related genes. Coculture of quail SSCs with mouse embryonic fibroblasts and Sertoli cells as a feeder layer resulted in the generation of stable SSC colonies and short-term cultivation, and the expression of SSC and germ cell markers was maintained during several passages of culture. Collectively, these results demonstrate the efficient isolation and characterization of quail SSCs and the suitability of Sertoli cells as a feeder layer for in vitro culture of quail SSCs. Quail SSCs will facilitate the production of germline chimeras and transgenesis.

Perspectives on avian stem cells for poultry breeding.

Stem cells have prulipotency to differentiate into many types of cell lineages. Recent progress of avian biotechnology enabled us to analyze the developmental fate of the stem cells: embryonic stem cells / primordial germ cells (PGCs). The stem cells were identified in the central area of the area pellucida of the stage X blastoderms. These cells could be applied for production of germline chimeras and organ regeneration. Generation of medical substrate in transgenic chickens has considerable interests in pharmaceuticals. Sex alteration of the offspring should be enormously beneficial to the poultry industry. Fertilization of the sex‐reversed sperm could lead to sexual alteration of the offspring. These strategies using stem cells / PGCs should be one of the most powerful tools for future poultry breeding.

Dnd Is a Critical Specifier of Primordial Germ Cells in the Medaka Fish.

SummaryPrimordial germ cell (PGC) specification occurs early in development. PGC specifiers have been identified in Drosophila, mouse, and human but remained elusive in most animals. Here we identify the RNA-binding protein Dnd as a critical PGC specifier in the medaka fish (Oryzias latipes). Dnd depletion specifically abolished PGCs, and its overexpression boosted PGCs. We established a single-cell culture procedure enabling lineage tracing in vitro. We show that individual blastomeres from cleavage embryos at the 32- and 64-cell stages are capable of PGC production in culture. Importantly, Dnd overexpression increases PGCs via increasing PGC precursors. Strikingly, dnd RNA forms prominent particles that segregate asymmetrically. Dnd concentrates in germ plasm and stabilizes germ plasm RNA. Therefore, Dnd is a critical specifier of fish PGCs and utilizes particle partition as a previously unidentified mechanism for asymmetric segregation. These findings offer insights into PGC specification and manipulation in medaka as a lower vertebrate model.

Germline Modification and Engineering in Avian Species.

Production of genome-edited animals using germline-competent cells and genetic modification tools has provided opportunities for investigation of biological mechanisms in various organisms. The recently reported programmed genome editing technology that can induce gene modification at a target locus in an efficient and precise manner facilitates establishment of animal models. In this regard, the demand for genome-edited avian species, which are some of the most suitable model animals due to their unique embryonic development, has also increased. Furthermore, germline chimera production through long-term culture of chicken primordial germ cells (PGCs) has facilitated research on production of genome-edited chickens. Thus, use of avian germline modification is promising for development of novel avian models for research of disease control and various biological mechanisms. Here, we discuss recent progress in genome modification technology in avian species and its applications and future strategies.

Production of Interspecific Germline Chimeras via Embryo Replacement.

In avian species, primordial germ cells (PGCs) use the vascular system to reach their destination, the genital ridge. Because of this unique migratory route of avian germ cells, germline chimera production can be achieved via germ cell transfer into a blood vessel. This study was performed to establish an alternative germ cell-transfer system for producing germline chimeras by replacing an original host embryo with a donor embryo, while retaining the host extraembryonic tissue and yolk, before circulation. First, to test the migratory capacity of PGCs after embryo replacement, Korean Oge (KO) chick embryos were used to replace GFP transgenic chick embryos. Four days after replacement, GFP-positive cells were detected in the replaced KO embryonic gonads, and genomic DNA PCR analysis with the embryonic gonads demonstrated the presence of the GFP transgene. To produce an interspecific germline chimera, the original chick embryo proper was replaced with a quail embryo onto the chick yolk. To detect the gonadal PGCs in the 5.5-day-old embryonic gonads, immunohistochemistry was performed with monoclonal antibodies specific to either quail or chick PGCs, i.e., QCR1 and anti-stage-specific embryonic antigen-1 (SSEA-1), respectively. Both the QCR1-positive and SSEA-1-positive cells were detected in the gonads of replaced quail embryos. Forty percent of the PGC population in the quail embryos was occupied by chick extraembryonically derived PGCs. In conclusion, replacement of an embryo onto the host yolk before circulation can be applied to produce interspecies germline chimeras, and this germ cell-transfer technology is potentially applicable for reproduction of wild or endangered bird species.

Generation of Efficient Germ-Line Chimeras Using Embryonic Stem Cell Injection.

There are many different reasons for producing germ-line chimeras, so a method for producing these is very important both for the testing of stem cells (SC) and for the production of an animal which may be genetically modified (Voncken, Methods Mol Biol 693:11-36, 2011). As with many scientific procedures the theory behind the process is very simple: in this case injection of cells into the blastocoel cavity of an embryo which has developed to the blastocyst stage so as the injected cells can contribute to the inner cell mass (ICM) and hopefully contribute to the germ line of the animal produced (Schneider et al., Stem Cell Rev 5(4):369-377, 2009). Incorporation of the cells into the gonads of the animal produced will allow the testing of those cells and the resulting animal which may be derived from the injected cells (Bradley et al., Nature 309(5965):255-256, 1984). The problems arise because of the size of the cells and the challenge of injection into the blastocoel cavity of a developing embryo.

한국재래닭(오계)의 원시 생식 세포의 냉동 보존에 있어서 여러 조건의 평가

동결 닭 원시 생식 세포의 생식계열 키메라를 이용한 생체에의 복원을 실용화 하기 위해서는, 닭 원시 생식 세포의 동결 보존 기술의 향상에 의해 동결 및 융해 후의 많은 생존세포를 확보하는 것이 반드시 필요하다. 닭 원시 생식 세포는 배양 5.5일령의 닭 원시 생식선으로부터 채취하고, MACS 방법에 의해서 순수 닭 원시 생식 세포를 분리했다. 15% 각각의 EG를 동결 보호제로 사용한 처리군이 각 군의 농도에 상관없이 유의적(p<0.05)으로 PG 처리군보다 동결 및 융해 후의 세포의 생존율이 높음을 확인하였다. 특히, 동결 보호제로 10% EG를 이용한 유리화 처리군에서 85.63%로 동일한 농도의 PG 처리군(66.81%)보다 유의적(p<0.05)으로 가장 높은 생존율을 보였다. 한편, 10% EG를 이용한 완만 동결 처리군에서 66.14%로 동일한 농도의 PG 처리군(50.11%)보다 유의적(p<0.05)으로 가장 높은 생존율을 보였다. 이상의 결과들로부터 유리화 동결에 있어서 가장 높은 생존율을 보인 10% EG이 최적의 동결 보호제로서 사용 가능함을 확인하였고, 이는 한국재래닭(오계)의 원시 생식 세포의 동결 보존의 실용화가 보다 더 향상될 수 있는 또 하나의 방법이 될 수 있음을 시사한다. Cryopreserving cells which are maintaining their viability are the very complex process. This study has been carried out in order to find the effects of cryopreservation steps and freezing media on the rates of viability of cryopreserved chicken primordial germ cells (PGCs). PGCs obtained from the germinal gonade of 5.5~6 day (stage 28) chick embryos of Korean Ogye (KO) and Commercial breeds (C), using the MACS method were suspended in a freezing medium containing a freezing and protecting agents (e.g. dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG)). Gonads were harvested from stage 28 chick embryos and pooled in groups of 5, 10, 15, 20E embryos, contributing gonads to the cell suspension. The gonadal cells, including PGCs, were then frozen in 1 of the following cryoprotectant treatments : 2.5%, 5%, 10%, 15% and 0% cryoprotectant (DMSO, EG, PG) as a control. Effects of exposure to slow freezing and vitrification, with different concentrations of the cryoprotectant solution, were examined. After vitrification and slow freezing, survival rates of the frozen-thawed PGCs from the 10% EG plus FBS treatment were 85.63%, and 66.14% (p<0.05), respectively. The viability of PGCs after freeze-thawing was significantly higher for 10% EG plus FBS treatment than for 10% PG + FBS treatment (p<0.05) (85.63% vs 66.81%) by vitrification. This study established a method for preserving chicken PGCs that enables systematic storage and labeling of cryopreserved PGCs in liquid (<TEX>$LN_2$</TEX>) at a germplasm repository and ease of entry into a data base. In the future, the importance for this new technology is that poultry lines can be conserved while work is being conducted on improving the production of germline chimeras.