Chicken Primordial Germ Cells Research Articles

Chicken blastoderms and primordial germ cells possess a higher expression of DNA repair genes and lower expression of apoptosis genes to preserve their genome stability

DNA is susceptible to damage by various sources. When the DNA is damaged, the cell repairs the damage through an appropriate DNA repair pathway. When the cell fails to repair DNA damage, apoptosis is initiated. Although several genes are involved in five major DNA repair pathways and two major apoptosis pathways, a comprehensive understanding of those gene expression is not well-understood in chicken tissues. We performed whole-transcriptome sequencing (WTS) analysis in the chicken embryonic fibroblasts (CEFs), stage X blastoderms, and primordial germ cells (PGCs) to uncover this deficiency. Stage X blastoderms mostly consist of undifferentiated progenitor (pluripotent) cells that have the potency to differentiate into all cell types. PGCs are also undifferentiated progenitor cells that later differentiate into male and female germ cells. CEFs are differentiated and abundant somatic cells. Through WTS analysis, we identified that the DNA repair pathway genes were expressed more highly in blastoderms and high in PGCs than CEFs. Besides, the apoptosis pathway genes were expressed low in blastoderms and PGCs than CEFs. We have also examined the WTS-based expression profiling of candidate pluripotency regulating genes due to the conserved properties of blastoderms and PGCs. In the results, a limited number of pluripotency genes, especially the core transcriptional network, were detected higher in both blastoderms and PGCs than CEFs. Next, we treated the CEFs, blastoderm cells, and PGCs with hydrogen peroxide (H2O2) for 1 h to induce DNA damage. Then, the H2O2 treated cells were incubated in fresh media for 3–12 h to observe DNA repair. Subsequent analyses in treated cells found that blastoderm cells and PGCs were more likely to undergo apoptosis along with the loss of pluripotency and less likely to undergo DNA repair, contrasting with CEFs. These properties of blastoderms and PGCs should be necessary to preserve genome stability during the development of early embryos and germ cells, respectively.

Targeted Knock-in of a Fluorescent Protein Gene into the Chicken Vasa Homolog Locus of Chicken Primordial Germ Cells using CRIS-PITCh Method.

In chickens, primordial germ cells (PGCs) are effective targets for advanced genome editing, including gene knock-in. Although a long-term culture system has been established for chicken PGCs, it is necessary to select a gene-editing tool that is efficient and precise for editing the PGC genome while maintaining its ability to contribute to the reproductive system. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and CRISPR-mediated precise integration into the target chromosome (CRIS-PITCh) methods are superior as the donor vector is easier to construct, has high genome editing efficiency, and does not select target cells, compared to the homologous recombination method, which has been conventionally used to generate knock-in chickens. In this study, we engineered knock-in chicken PGCs by integrating a fluorescent protein gene cassette as a fusion protein into the chicken vasa homolog (CVH) locus of chicken PGCs using the CRIS-PITCh method. The knock-in PGCs expressed the fluorescent protein in vitro and in vivo, facilitating the tracking of PGCs. Furthermore, we characterized the efficiency of engineering double knock-in cell lines. Knock-in cell clones were obtained by limiting dilution, and the efficiency of engineering double knock-in cell lines was confirmed by genotyping. We found that 82% of the analyzed clones were successfully knocked-in into both alleles. We suggest that the production of model chicken from the knock-in PGCs can contribute to various studies, such as the elucidation of the fate of germ cells and sex determination in chicken.

Finer resolution analysis of transcriptional programming during the active migration of chicken primordial germ cells

Primordial germ cells (PGCs) in chickens polarize and move passively toward the anterior region by the morphogenetic movement of the embryo. Further migration of PGCs towards the genital ridge via the germinal crescent region and blood vessels occurs actively through the chemoattractive signals. The mechanisms of initiation of PGCs migration, lodging the PGCs in the vascular system, and colonization of PGCs in the gonads are well-studied. However, transcriptome sequencing-based cues directing the migration of the PGCs towards gonads, some of the relevant molecules, biological processes, and transcription factors (TFs) are less studied in chickens. The current study comprehensively interprets the transcriptional programming of PGCs during their active migration (E2.5 to E8). Current results revealed several vital understandings, including a set of genes that upregulated male-specifically (XPA, GNG10, RPL17, RPS23, and NDUFS4) or female-specifically (HINTW, NIPBL, TERAL2, ATP5F1AW, and SMAD2W) in migrating PGCs, and transcriptionally distinct PGCs, particularly in the gonadal environment. We identified DNA methylation and histone modification-associated genes that are novel in chicken PGCs and show a time-dependent enrichment in migrating PGCs. We further identified a large number of differentially expressed genes (DEGs, including TFs) in blood PGCs (at E2.5) compared to gonadal PGCs (at E8) in both sexes; however, this difference was greater in males. We also revealed the enriched biological processes and signaling pathways of significant DEGs identified commonly, male-specifically, or female-specifically between the PGCs isolated at E2.5, E6, and E8. Collectively, these analyses provide molecular insights into chicken PGCs during their active migration phase.

Effect of miR-302b MicroRNA Inhibition on Chicken Primordial Germ Cell Proliferation and Apoptosis Rate.

The primordial germ cells (PGCs) are the precursors for both the oocytes and spermatogonia. Recently, a novel culture system was established for chicken PGCs, isolated from embryonic blood. The possibility of PGC long-term cultivation issues a new advance in germ cell preservation, biotechnology, and cell biology. We investigated the consequence of gga-miR-302b-5P (5P), gga-miR-302b-3P (3P) and dual inhibition (5P/3P) in two male and two female chicken PGC lines. In treated and control cell cultures, the cell number was calculated every four hours for three days by the XLS Imaging system. Comparing the cell number of control and treated lines on the first day, we found that male lines had a higher proliferation rate independently from the treatments. Compared to the untreated ones, the proliferation rate and the number of apoptotic cells were considerably reduced at gga-miR-302b-5P inhibition in all PGC lines on the third day of the cultivation. The control PGC lines showed a significantly higher proliferation rate than 3P inhibited lines on Day 3 in all PGC lines. Dual inhibition of gga-miR-302b mature miRNAs caused a slight reduction in proliferation rate, but the number of apoptotic cells increased dramatically. The information gathered by examining the factors affecting cell proliferation of PGCs can lead to new data in stem cell biology.

Knock-Out of Retrovirus Receptor Gene Tva in the Chicken Confers Resistance to Avian Leukosis Virus Subgroups A and K and Affects Cobalamin (Vitamin B12)-Dependent Level of Methylmalonic Acid.

The chicken Tva cell surface protein, a member of the low-density lipoprotein receptor family, has been identified as an entry receptor for avian leukosis virus of classic subgroup A and newly emerging subgroup K. Because both viruses represent an important concern for the poultry industry, we introduced a frame-shifting deletion into the chicken tva locus with the aim of knocking-out Tva expression and creating a virus-resistant chicken line. The tva knock-out was prepared by CRISPR/Cas9 gene editing in chicken primordial germ cells and orthotopic transplantation of edited cells into the testes of sterilized recipient roosters. The resulting tva −/− chickens tested fully resistant to avian leukosis virus subgroups A and K, both in in vitro and in vivo assays, in contrast to their susceptible tva +/+ and tva +/− siblings. We also found a specific disorder of the cobalamin/vitamin B12 metabolism in the tva knock-out chickens, which is in accordance with the recently recognized physiological function of Tva as a receptor for cobalamin in complex with transcobalamin transporter. Last but not least, we bring a new example of the de novo resistance created by CRISPR/Cas9 editing of pathogen dependence genes in farm animals and, furthermore, a new example of gene editing in chicken.

Dual regulatory actions of LncBMP4 on BMP4 promote chicken primordial germ cell formation.

The unique characteristics of chicken primordial germ cells (PGCs) provide potential strategies for transgenic animal generation; however, insufficient PGC availability has limited their application. Regulation of bone morphogenic protein 4 (BMP4), a crucial factor for PGCs formation, may provide new strategies for PGC generation. We here identify a long noncoding RNA (lncRNA) that targets BMP4 (LncBMP4). LncBMP4 has similar functions as BMP4, in that it facilitates the formation and migration of PGCs. LncBMP4 promotes BMP4 expression by adsorbing the miRNA gga-mir-12211, thus reducing its inhibitory effect on BMP4 expression. In addition, the small peptide EPC5 encoded by LncBMP4 promotes the transcription of BMP4. The competing endogenous RNA (ceRNA) effect of LncBMP4 requires N6-methyladenosine (m6A) modification, in a dose-dependent manner, and high levels of m6A modification hinder EPC5 translation. Understanding the molecular mechanisms through which LncBMP4 promotes BMP4 expression during PGC formation may provide new avenues for efficient PGC generation.

Effect of Glutamine Stability on the Long-term Culture and Line Establishment of Chicken Primordial Germ Cells

Primordial germ cells (PGCs) are precursors for mature gametes, which transmit genetic information to the next generation. Due to the importance of PGCs in many fields including developmental biology, genome editing, transgenesis, and conservation of avian genetic resources, a lot of research has focused on the cultivation of PGCs. Despite considerable progress in the establishment of specified culture media for the expansion of PGCs, a well-defined PGC culture medium has not yet been developed. This might be due to the complexity of the nutritional requirements of PGCs in the culture. Besides the nutritional needs including vitamins, amino acids, salts, carbohydrates, and growth factors, a certain source of energy must be provided to sustain growth and viability. Glutamine is a major energy source for cultured cells, which is commonly added in cell culture media at higher concentrations than other amino acids. However, glutamine is very labile and rapidly degrades in solutions such as culture media. This generates ammonia as a by-product, which is toxic to the cultured cells and can affect cell viability and protein glycosylation. Therefore, the stability of glutamine in culture conditions is another concern for the long-termed culture of PGCs. Here, we study the effect of glutamine stability on PGC culture using glutamine and GlutaMax (a commercial stabilized dipeptide form of glutamine). We found that the addition of GlutaMax in the medium promotes PGC proliferation. This effect might be exerted by minimizing toxic ammonia buildup that results in maximizing cell performance, and also increasing media stability.

Cultivation and characterization of primordial germ cells from blue layer hybrids (Araucana crossbreeds) and generation of germline chimeric chickens

The chicken (Gallus gallus) is one of the most common and widespread domestic species, with an estimated total population of 25 billion birds worldwide. The vast majority of chickens in agriculture originate from hybrid breeding programs and is concentrated on few commercially used high performance lines, whereas numerous local and indigenous breeds are at risk to become extinct. To preserve the genomic resources of rare and endangered chicken breeds innovative methods are necessary. Here, we established a solid workflow for the derivation and biobanking of chicken primordial germ cells (PGCs) from blue layer hybrids. To achieve this, embryos of a cross of heterozygous blue egg layers were sampled to obtain blood derived and gonadal male as well as female PGCs of different genotypes (homozygous, heterozygous and nullizygous blue-allele bearing). The total efficiency of established PGC lines was 45% (47/104) within an average of 49 days until they reached sufficient numbers of cells for cryopreservation. The stem-cell character of the cultivated PGCs was confirmed by SSEA-1 immunostaining, and RT-PCR amplification of the pluripotency- and PGC-specific genes cPOUV, cNANOG, cDAZL and CVH. The Sleeping Beauty transposon system allowed to generate a stable integration of a Venus fluorophore reporter into the chicken genome. Finally, we demonstrated that, after re-transfer into chicken embryos, Venus-positive PGCs migrated and colonized the forming gonads. Semen samples of 13 raised cell chimeric roosters were analyzed by flow cytometry for the efficiency of germline colonization by the transferred PGCs carrying the Venus reporter and their proper differentiation into vital spermatids. Thus, we provide a proof-of-concept study for the potential use of PGCs for the cryobanking of rare breeds or rare alleles.

PiggyBac Transposition and the Expression of Human Cystatin C in Transgenic Chickens.

Simple SummaryThe genetic modification of livestock genomes showed the great potential for production of industrial biomaterials as well as improving animal production. Particularly, the transgenic hen’s eggs have been considered for a massive production system of the genetically engineered biomaterials as a bioreactor animal. Virus-mediated transgene transduction is the most powerful strategy to generate the transgenic animals. However, industrial applications were hampered by many obstacles such as relatively low germline transmission and transgene silencing effects, as well as viral safety issues. In this study, a piggyBac transposon which is a non-viral integration technical platform was introduced into chicken primordial germ cells. Finally, we developed transgenic chickens and assayed the bioactivity of human cystatin C in the transgenic chicken’s tissues.A bioreactor can be used for mass production of therapeutic proteins and other bioactive substances. Although various methods have been developed using microorganisms and animal cells, advanced strategies are needed for the efficient production of biofunctional proteins. In microorganisms, post-translational glycosylation and modification are not performed properly, while animal cell systems require more time and expense. To overcome these problems, new methods using products from transgenic animals have been considered, such as genetically modified cow’s milk and hen’s eggs. In this study, based on a non-viral piggyBac transposition system, we generated transgenic bioreactor chickens that produced human cystatin C (hCST3). There were no differences in the phenotype or histochemical structure of the wild-type and hCST3-expressing transgenic chickens. Subsequently, we analyzed the hCST3 expression in transgenic chickens, mainly in muscle and egg white, which could be major deposition warehouses for hCST3 protein. In both muscle and egg white, we detected high hCST3 expression by ELISA and Western blotting. hCST3 proteins were efficiently purified from muscle and egg white of transgenic chickens using a His-tag purification system. These data show that transgenic chickens can be efficiently used as a bioreactor for the mass production of bioactive materials.

Development of optimal technological approaches for obtaining PGCs in Pushkin breed chickens for further transformation by the CRISPR / Cas9 system

The CRISPR / Cas9 genome editing system is a very convenient and promising method for creating highly productive chickens of meat direction with improved productive qualities. The biological feature of the amniotes structure creates certain difficulties for the an editing structure introduction into a unicellular zygote. The aim of the study is to optimize technological approaches for obtaining PGCs (Primordial Germ Cells) in Pushkin chickens from the Bioresource collection RRIFAGB “Genetic collection of rare and endangered chicken breeds” (Pushkin, St. Petersburg) for further transformation by the CRISPR / Cas9 system. All experiments on chickens were conducted with the ethical approval of the Russian Research Institute of Farm Animal Genetics and Breeding - Branch of the L.K. Ernst Federal Research Center for Animal Husbandry (Protocol Number: 2020–3). The optimal egg weight varied from 55 to 62 grams for incubation It has been revealed that embryos obtained from large eggs from 62 grams or more developed poorly or did not reach the stage of PGCs selection at all. The eggs were incubated with occasional shaking at 37–38 ° C and 65–70% relative humidity. It was better to select PGCs of Pushkin breed chickens on the 3rd day of incubation from the dorsal aorta of embryos (Hamburger Hamilton stage 14). Cell selection was performed using a microinjector (Narishige IM-11-2, Japan). The cells were cultured for 21 days at 37 ° C with 5% CO2. PGCs have been found to reproduce better in a serum-free medium (MEM Non-Essential Amino Acids Solution, Gibco, Thermo Fisher) of a specific chemical composition than in a medium containing serum (Opti-MEM with Chicken Serum, Gibco, Thermo Fisher). The expression of PGCs various genes characteristic was determined by real-time PCR using conservative primer sets.

Optimization of transfection conditions of chicken primordial germ cells.

To improve the transfection efficiency of chicken primordial germ cells (PGCs), the present study evaluated the plasmid dosage and cell number on the efficiencies of three transfection reagents (Lipofectamine 2000, 3000 and LTX & Plus Reagent). PGCs was isolated from embryonic gonads of Huiyang bearded chicken. After 60 days of culture in vitro, the cells were transfected by using Lipofectamine transfection reagents with piggyBac vectors coding for the green fluorescence protein (GFP). PGCs were passaged in culture and fluorescent cells were screened and selected by flow cytometry at three days after transfection. At three weeks post transfection, about 2000 cells were injected into the stage 16 Hamburger and Hamilton (HH) embryos and incubated until stage 30 HH. The results showed that Lipofectamine 3000 was the best for transfection of PGCs. The highest transfection efficiency of PGCs could be achieved with a combination of 3 μg plasmid, 4 μL Lipofectamine 3000 transfection reagent and 0.5×10 4PGCs cells. Flow cytometry analysis showed a 23.4% efficiency of stable transfection of PGCs using Lipofectamine 3000 with piggyBac vector, which was improved 2 times or more over current commonly used methods. After reinjecting PGCs into recipient chicken embryos, GFP-positive cells were observed in the gonads of the recipient chicken embryo by fluorescence microscopy. The study comprehensively evaluated the factors of transfection reagents, plasmid dosage and cell number to optimize the transfection of PGCs, thereby providing a foundation for the efficient preparation of transgenic and gene-edited chickens.

Differential transcriptional regulation of the NANOG gene in chicken primordial germ cells and embryonic stem cells

BackgroundNANOG is a core transcription factor (TF) in embryonic stem cells (ESCs) and primordial germ cells (PGCs). Regulation of the NANOG gene by TFs, epigenetic factors, and autoregulatory factors is well characterized in ESCs, and transcriptional regulation of NANOG is well established in these cells. Although NANOG plays a key role in germ cells, the molecular mechanism underlying its transcriptional regulation in PGCs has not been studied. Therefore, we investigated the mechanism that regulates transcription of the chicken NANOG (cNANOG) gene in PGCs and ESCs.ResultsWe first identified the transcription start site of cNANOG by 5′-rapid amplification of cDNA ends PCR analysis. Then, we measured the promoter activity of various 5′ flanking regions of cNANOG in chicken PGCs and ESCs using the luciferase reporter assay. cNANOG expression required transcriptional regulatory elements, which were positively regulated by POU5F3 (OCT4) and SOX2 and negatively regulated by TP53 in PGCs. The proximal region of the cNANOG promoter contains a positive transcriptional regulatory element (CCAAT/enhancer-binding protein (CEBP)-binding site) in ESCs. Furthermore, small interfering RNA-mediated knockdown demonstrated that POU5F3, SOX2, and CEBP played a role in cell type-specific transcription of cNANOG.ConclusionsWe show for the first time that different trans-regulatory elements control transcription of cNANOG in a cell type-specific manner. This finding might help to elucidate the mechanism that regulates cNANOG expression in PGCs and ESCs.

Marker counter-selection via CRISPR/Cas9 co-targeting for efficient generation of genome edited avian cell lines and germ cells

Efficient isolation of genetically modified cells that are phenotypically indistinguishable from the unmodified cells remains a major technical barrier for the broader utilization of CRISPR/Cas9. Here, we report a novel enrichment approach to select the genome engineered cells by co-targeting a genomically integrated GFP gene along with the endogenous gene of interest (GOI). Using this co-targeting approach, multiple genomic loci were successfully targeted in chicken (DF1) and quail (CEC-32) fibroblast cell lines by transient transfection of Cas9 and guide RNAs (gRNAs). Clonal isolation of co-targeted DF1 cells showed 75% of cell clones had deletion of GFP and biallelic deletion of the GOI. To assess the utility of this approach to generate genome modified animals, we tested it on chicken primordial germ cells (PGCs) expressing GFP by co-targeting with gRNAs against GFP and endogenous ovomucoid (OVM) gene. PGCs enriched for loss of GFP and confirmed for OVM deletion, derived by co-targeting, were injected into Hamburger and Hamilton stage 14–15 chicken embryos, and their ability to migrate to the genital ridge was confirmed. This simple, efficient enrichment approach could easily be applied to the creation of knock-out or edited cell lines or animals.

Long Noncoding RNA LncPGCR Mediated by TCF7L2 Regulates Primordial Germ Cell Formation in Chickens.

Simple SummaryThe potential of primordial germ cells (PGCs) for multidirectional differentiation, together with their unique regeneration ability, makes them one of the most promising seed cells in clinical medicine and tissue engineering research. However, not enough PGCs can be obtained to meet the demand, which limits their application. We defined a novel long noncoding RNA (lncRNA) mediated by epigenetics, which could activate the miR-6577-5p/Btrc pathway to promote the formation of PGCs. The technical system we have established is a useful tool to obtain sufficient PGCs for scientific research. Our study offers great theoretical and practical value in the production of transgenic animals or genomic imprinting in poultry. We believe that our study will help researchers in the fields of agricultural production, developmental biology, and cell biology.Although lncRNAs have been identified as playing critical roles in the development of germ cells, their potential involvement in the development of PGCs in chickens remains poorly understood. Differentially expressed lncRNAs (DELs) from previous RNA-seq of embryonic stem cells (ESCs), PGCs, and spermatogonial stem cells (SSCs) were analyzed by K-means clustering, from which a key candidate, lncRNA (lncRNA PGC regulator, LncPGCR) was obtained. We confirmed that LncPGCR plays a positive role in the development of PGCs by increasing the expression of the PGC marker gene (Cvh and C-kit), while downregulating the pluripotency-associated gene (Nanog) in vitro and in vivo. The activation and expression of LncPGCR are regulated by histone acetylation, and transcription factor TCF7L2. Mechanistically, a rescue assay was performed to further confirm that LncPGCR contributed to the development of PGCs by regulating the gga-miR-6577-5p/Btrc signaling pathway. Adsorption of gga-miR-6577-5p activated the WNT signaling cascade by relieving the gga-miR-6577-5p-dependent inhibition of Btrc expression. Taken together, our study discovered the growth-expedited role of LncPGCR in PGCs development, showing the potential LncPGCR/miR-6577-5p/Btrc pathway. The results and findings provide a novel insight into the development of PGCs.

Study on the Function and Mechanism of Lin28B in the Formation of Chicken Primordial Germ Cells

Simple SummaryIn this study, we explored the function and molecular mechanism of Lin28B in the formation of chicken primordial germ cells (PGCs) in detail. Our results indicate that Lin28B participates in the formation of PGCs through let-7a-3p, which set a theoretical foundation for improving the function and mechanism of the Lin28 family in the formation of PGCs.Lin28A and Lin28B are two homologues of the same family of RNA binding proteins (RBPs). The function and molecular mechanism of Lin28A in the formation of primordial germ cells (PGCs) are very clear, but the related research on Lin28B is rarely reported. Here, we found that the overexpression of Lin28B can promote the formation of PGC in vivo. Furthermore, the overexpression of Lin28B also resulted in the inhibition of totipotency gene expression and upregulated the PGCs marker genes, and a significant increase in the number of PGCs in genital ridge, as detected by Periodic Acid-Schiff(PAS) staining. However, the inhibited Lin28B expression showed completely opposite results, which were confirmed on the PGC induction model in vitro. Mechanistically, we found that the overexpression of Lin28B can inhibit the maturation of let-7a-3p, and the results of high-throughput sequencing indicated that let-7a-3p was a negative regulator of the formation process of PGCs. Therefore, we conclude that our results determine that Lin28B participates in the formation of PGCs through let-7a-3p, which set a theoretical foundation for improving the function and mechanism of Lin28 family in the formation of PGCs.

Transcriptome Sequencing and Comparative Analysis of Amphoteric ESCs and PGCs in Chicken (Gallus gallus).

Simple SummaryThe study of chicken embryonic stem cells (ESCs) and primordial germ cells (PGCs) showed the potential application of developmental biology and translational medicine. However, the difference between amphoteric ESCs and PGCs is still elusive, limiting the accuracy of correlative research. In this paper, chicken amphoteric ESCs and PGCs were isolated, separated, and sequenced to explore their dynamic transcriptomes. Our results provide a knowledge base of transcriptomes in chicken amphoteric ESCs and PGCs, which will help other researchers interested in studying relative biological processes.Chicken (Gallus gallus) pluripotent embryonic stem cells (ESCs) and primordial germ cells (PGCs) can be broadly applied in the research of developmental and embryonic biology, but the difference between amphoteric ESCs and PGCs is still elusive. This study determined the sex of collected samples by identifying specific sex markers via polymerase chain reaction (PCR) and fluorescence activated cell sorter (FACS). RNA-seq was utilized to investigate the transcriptomic profile of amphoteric ESCs and PGCs in chicken. The results showed no significant differentially expressed genes (DEGs) in amphoteric ESCs and 227 DEGs exhibited in amphoteric PGCs. Moreover, those 227 DEGs were mainly enriched in 17 gene ontology (GO) terms and 27 pathways according to Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Furthermore, qRT-PCR was performed to verify RNA-seq results, and the results demonstrated that Notch1 was highly expressed in male PGCs. In summary, our results provided a knowledge base of chicken amphoteric ESCs and PGCs, which is helpful for future research in relevant biological processes.

Highly elevated base excision repair pathway in primordial germ cells causes low base editing activity in chickens.

Base editing technology enables the generation of precisely genome-modified animal models. In this study, we applied base editing to chicken, an important livestock animal in the fields of agriculture, nutrition, and research through primordial germ cell (PGC)-mediated germline transmission. Using this approach, we successfully produced two genome-modified chicken lines harboring mutations in the genes encoding ovotransferrin (TF) and myostatin (MSTN); however, only 55.5% and 35.7% of genome-modified chickens had the desired base substitutions in TF and MSTN, respectively. To explain the low base-editing activity, we performed molecular analysis to compare DNA repair pathways between PGCs and the chicken fibroblast cell line DF-1. The results revealed that base excision repair (BER)-related genes were significantly elevated in PGCs relative to DF-1 cells. Subsequent functional studies confirmed that the editing activity could be regulated by modulating the expression of uracil N-glycosylase (UNG), an upstream gene of the BER pathway. Collectively, our findings indicate that the distinct DNA repair property of chicken PGCs causes low editing activity during genome modification, however, modulation of BER functions could promote the production of genome-modified organisms with the desired genotypes.

In Vitro Culture of Chicken Circulating and Gonadal Primordial Germ Cells on a Somatic Feeder Layer of Avian Origin.

Simple SummaryChicken primordial germ cells are specialized cells that are formed outside the developing embryo, from where they migrate into the gonad, and give rise to the gametes. There are two types of those cells: Circulating blood primordial germ cells and gonadal primordial germ cells. They can be isolated only in low numbers from the bloodstream or gonads of donor embryos. Hence, efficient in vitro cultivation systems are required to increase their quantity through proliferation. Here, we provide a single culture system that can be used to cultivate both cell types. We also present a novel, easy-to-train, and non-invasive method to identify live primordial germ cells in the culture. In this analysis, chicken primordial germ cells obtained from embryonic blood or gonadal regions were cultured in vitro on feeder cell layers derived from embryonic chick cells. The use of the chicken origin feeder layer allowed reducing the xenogenic animal factors in the culture. We demonstrated a feasible and cost-efficient technique to routinely assess the cultivated primordial germ cells on the basis of their morphological characteristics and using the optical features of cells in darkfield illumination. This method is especially useful to distinguish primordial germ cells during co-cultivation with other cell types.The present study had two aims: (1) To develop a culture system that imitates a normal physiological environment of primordial germ cells (PGCs). There are two types of PGCs in chicken: Circulating blood (cPGCs) and gonadal (gPGCs). The culture condition must support the proliferation of both cPGCs and gPGCs, without affecting their migratory properties and must be deprived of xenobiotic factors, and (2) to propose an easy-to-train, nonlabeling optical technique for the routine identification of live PGCs. To address the first aim, early chicken embryo’s feeder cells were examined instead of using feeder cells from mammalian species. The KAv-1 medium at pH 8.0 with the addition of bFGF (basic fibroblast growth factor) was used instead of a conventional culture medium (pH approximately 7.2). Both cPGCs and gPGCs proliferated in vitro and retained their migratory ability after 2 weeks of culture. The cultivated cPGCs and gPGCs colonized the right and/or left gonads of the recipient male and female embryos. To address the second aim, we demonstrated a simple and rapid method to identify live PGCs as bright cells under darkfield illumination. The PGCs rich in lipid droplets in their cytoplasm highly contrasted with the co-cultured feeder layer and other cell populations in the culture.

Concentration and total number of circulating primordial germ cells in Green-legged Partridgelike chicken embryos

The Green-legged Partridgelike fowl is an old Polish indigenous breed of chicken. Primordial germ cells (PGCs) are one of the best sources of precursor cells that can be used for the conservation and proliferation of the endangered breeds of bird. Initially, the chicken PGCs colonize at the anterior extraembryonic region called “germinal crescent,” and after the establishment of blood vascular circulation, they temporally circulate via the embryonic blood vascular system along with embryonic blood cells. They further colonize at the microcapillary networks of both right and left future gonadal regions. Subsequently, they migrate interstitially to reach gonadal anlages, where they begin to differentiate and eventually develop into the future ova or sperm. The basic knowledge regarding the concentration and the total number of circulating PGCs (cPGCs) throughout their circulating phase in the early embryonic stages is crucial for providing an insight into the mechanisms by which they circulate and colonize at the capillary networks of left and right future gonadal regions in each developmental stage. The present study aims to determine the most efficient developmental stage that is suitable to collect cPGCs. The concentration of cPGCs was directly measured, and total volume of embryonic blood was calculated based on the concentration of PKH26-stained embryonic blood cells which were injected 10 min before the blood sampling process in the same embryo during each stage of embryonic development from stage 13 Hamburger and Hamilton (HH; Hamburger and Hamilton, 1951) to 16 HH. Analysis of whole embryonic bloodstream revealed that at stage 14 HH of embryonic development, peak total number of cPGCs (386.3 cells/μL) and peak concentration of cPGCs (18.6 cells/μL) were observed. Later, there was a decrease in concentration, suggesting that the cPGCs might be trapped gradually by the capillary networks at the future gonadal regions after stage 15 HH.

Narrow H3K4me2 is required for chicken PGC formation.

The development of primordial germ cells (PGCs) undergoes epigenetic modifications. The study of histone methylation in regulating PGCs is beneficial to understand the development and differentiation mechanism of germ stem cells. Notably, it provides a theoretical basis for directed induction and mass acquisition in vitro. However, little is known about the regulation of PGC formation by histone methylation. Here, we found the high enrichment of H3K4me2 in the blastoderm, genital ridges, and testis. Chromatin immunoprecipitation sequencing was performed and the results revealed that genomic H3K4me2 is dynamic in embryonic stem cells, PGCs, and spermatogonial stem cells. This trend was consistent with the H3K4me2 enrichment in the gene promoter region. Additionally, narrow region triggered PGC-related genes (Bmp4, Wnt5a, and Tcf7l2) and signaling pathways (Wnt and transforming growth factor-β). After knocking down histone methylase Mll2 in vitro and vivo, the level of H3K4me2 decreased, inhibiting Cvh and Blimp1 expression, then repressing the formation of PGCs. Taken together, our study revealed the whole genome map of H3K4me2 in the formation of PGCs, contributing to improve the epigenetic study in PGC formation and providing materials for bird gene editing and rescue of endangered birds.