Identification of a novel risk factor for chronic wasting disease (CWD) in elk: S100G single nucleotide polymorphism (SNP) of the prion protein gene (PRNP).

Prion diseases are fatal and malignant infectious encephalopathies induced by the pathogenic form of prion protein (PrPSc) originating from benign prion protein (PrPC). A previous study reported that the M132L single nucleotide polymorphism (SNP) of the prion protein gene (PRNP) is associated with susceptibility to chronic wasting disease (CWD) in elk. However, a recent meta-analysis integrated previous studies that did not find an association between the M132L SNP and susceptibility to CWD. Thus, there is controversy about the effect of M132L SNP on susceptibility to CWD. In the present study, we investigated novel risk factors for CWD in elk. We investigated genetic polymorphisms of the PRNP gene by amplicon sequencing and compared genotype, allele, and haplotype frequencies between CWD-positive and CWD-negative elk. In addition, we performed a linkage disequilibrium (LD) analysis by the Haploview version 4.2 program. Furthermore, we evaluated the 3D structure and electrostatic potential of elk prion protein (PrP) according to the S100G SNP using AlphaFold and the Swiss-PdbViewer 4.1 program. Finally, we analyzed the free energy change of elk PrP according to the S100G SNP using I-mutant 3.0 and CUPSAT. We identified 23 novel SNP of the elk PRNP gene in 248 elk. We found a strong association between PRNP SNP and susceptibility to CWD in elk. Among those SNP, S100G is the only non-synonymous SNP. We identified that S100G is predicted to change the electrostatic potential and free energy of elk PrP. To the best of our knowledge, this was the first report of a novel risk factor, the S100G SNP, for CWD.

Efficient gene delivery into the embryonic chicken brain using neuron-specific promoters and in ovo electroporation.

BACKGROUND: The chicken in ovo model is an attractive system to explore underlying mechanisms of neural and brain development, and it is important to develop effective genetic modification techniques that permit analyses of gene functions in vivo. Although electroporation and viral vector-mediated gene delivery techniques have been used to introduce exogenous DNA into chicken embryonic cells, transducing neurons efficiently and specifically remains challenging. METHODS: In the present study, we performed a comparative study of the ubiquitous CMV promoter and three neuron-specific promoters, chicken Ca2+/calmodulin-dependent kinase (cCaMKII), chicken Nestin (cNestin), and human synapsin I. We explored the possibility of manipulating gene expression in chicken embryonic brain cells using in ovo electroporation with the selected promoters. RESULTS: Transgene expression by two neuron-specific promoters (cCaMKII and cNestin) was preliminarily verified in vitro in cultured brain cells, and in vivo, expression levels of an EGFP transgene in brain cells by neuron-specific promoters were comparable to or higher than those of the ubiquitous CMV promoter. Overexpression of the FOXP2 gene driven by the cNestin promoter in brain cells significantly affected expression levels of target genes, CNTNAP2 and ELAVL4. CONCLUSION: We demonstrated that exogenous DNA can be effectively introduced into neuronal cells in living embryos by in ovo electroporation with constructs containing neuron-specific promoters. In ovo electroporation offers an easier and more efficient way to manipulate gene expression during embryonic development, and this technique will be useful for neuron-targeted transgene expression.

DMRT1 gene disruption alone induces incomplete gonad feminization in chicken.

Compared with the well-described XY sex determination system in mammals, the avian ZW sex determination system is poorly understood. Knockdown and overexpression studies identified doublesex and mab-3-related transcription factor 1 (DMRT1) as the testis-determining gene in chicken. However, the detailed effects of DMRT1 gene disruption from embryonic to adult development are not clear. Herein, we have generated DMRT1-disrupted chickens using the clustered regularly interspaced short palindromic repeats-associated protein 9 system, followed by an analysis of physiological, hormonal, and molecular changes in the genome-modified chickens. In the early stages of male chicken development, disruption of DMRT1 induced gonad feminization with extensive physiological and molecular changes; however, functional feminine reproductivity could not be implemented with disturbed hormone synthesis. Subsequent RNA-sequencing analysis of the DMRT1-disrupted chicken gonads revealed gene networks, including several novel genes linearly and non-linearly associated with DMRT1, which are involved in gonad feminization. By comparing the gonads of wild type with the genome-modified chickens, a set of genes were identified that is involved in the ZW sex determination system independent of DMRT1. Our results extend beyond the Z-dosage hypothesis to provide further information about the avian ZW sex determination system and epigenetic effects of gonad feminization.

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.

Targeted gene insertion into Z chromosome of chicken primordial germ cells for avian sexing model development.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) have facilitated the production of genome-edited animals for use as models. Because of their unique developmental system, avian species offer many advantages as model vertebrates. Here, we report the development of novel chicken models using the CRISPR/Cas9-mediated nonhomologous end joining repair pathway in chicken primordial germ cells (PGCs). Through the introduction of a donor plasmid containing short guide RNA recognition sequences and CRISPR/Cas9 plasmids into chicken PGCs, exogenous genes of donor plasmids were precisely inserted into target loci, and production of transgenic chickens was accomplished through subsequent transplantation of the Z chromosome-targeted PGCs. Using this method, we successfully accomplished the targeted gene insertion to the chicken sex Z chromosome without detected off-target effects. The genome-modified chickens robustly expressed green fluorescent protein from the Z chromosome, which could then be used for easy sex identification during embryogenesis. Our results suggest that this powerful genome-editing method could be used to develop many chicken models and should significantly expand the application of genome-modified avians.-Lee, H. J., Yoon, J. W., Jung, K. M., Kim, Y. M., Park, J. S., Lee, K. Y., Park, K. J., Hwang, Y. S., Park, Y. H., Rengaraj, D., Han, J. Y. Targeted gene insertion into Z chromosome of chicken primordial germ cells for avian sexing model development.

Single-cell transcriptome analysis of male chicken germ cells reveals changes in signaling pathway-related gene expression profiles during mitotic arrest.

Mitotic arrest is necessary for the embryonic development of germ cells, and thus, it is important to understand the signaling pathways that regulate mitotic arrest. Here, we investigated the signaling pathway dynamics of male embryonic chicken germ cells during mitotic arrest by single-cell transcriptome analysis using germ-cell tracing models. We identified signaling pathways that change at the transcriptional level during chicken male germ-cell development after sex determination. We found that several components of the BMP, Notch, and JAK-STAT signaling pathways were downregulated at the mitotic-arrest stage and were reactivated 1 week after hatching when all germ cells are quiescent after entering mitotic arrest. In addition, the transcriptional levels of components of the MAPK, Hedgehog, and thyroid-hormone signaling pathways were steadily upregulated after mitotic arrest. This suggests the cooperation of multiple signaling pathways during entry into mitotic arrest and subsequent quiescence of chicken male germ cells.

Autoclave treatment fails to completely inactivate DLB alpha-synuclein seeding activity.

Synucleinopathies are characterized by the deposition of alpha-synuclein (α-syn) aggregates in brain tissue. Pathological α-syn aggregates propagate in a prion-like manner and display prion-like biochemical properties. Using RT-QuIC, we measured α-syn seeding activity from brains of Dementia with Lewy body (DLB) patients post autoclave. Here, we show that autoclaving at 121 °C removes one to two log10 of α-syn seeding activity but the remaining 50% seeding dose (SD50) is more than 107/mg tissue. DLB brain samples autoclaved at 132 °C still revealed an SD50 of approximately 106/mg tissue. Our data suggest that DLB α-syn seeds are incompletely inactivated by standard autoclave, thus highlighting the need for evaluating laboratory procedures that fully inactivate them.

Production of recombinant human IgG1 Fc with beneficial N-glycosylation pattern for anti-inflammatory activity using genome-edited chickens.

Intravenous immunoglobulin (IVIG) is a plasma-derived polyclonal IgG used for treatment of autoimmune diseases. Studies show that α-2,6 sialylation of the Fc improves anti-inflammatory activity. Also, afucosylation of the Fc efficiently blocks FcγRIIIA by increasing monovalent affinity to this receptor, which can be beneficial for treatment of refractory immune thrombocytopenia (ITP). Here, we generated genome-edited chickens that synthesize human IgG1 Fc in the liver and secrete α-2,6 sialylated and low-fucosylated human IgG1 Fc (rhIgG1 Fc) into serum and egg yolk. Also, rhIgG1 Fc has higher affinity for FcγRIIIA than commercial IVIG. Thus, rhIgG1 Fc efficiently inhibits immune complex-mediated FcγRIIIA crosslinking and subsequent ADCC response. Furthermore, rhIgG1 Fc exerts anti-inflammatory activity in a passive ITP model, demonstrating chicken liver derived rhIgG1 Fc successfully recapitulated efficacy of IVIG. These results show that genome-edited chickens can be used as a production platform for rhIgG1 Fc with beneficial N-glycosylation pattern for anti-inflammatory activities.

Sequential verification of exogenous protein production in OVA gene-targeted chicken bioreactors.

The chicken has potential as an efficient bioreactor system because of its outstanding protein production capacity and low cost. The CRISPR/Cas9-mediated gene-editing system enables production of highly marketable exogenous proteins in transgenic chicken bioreactors. However, because it takes approximately 18 mo to evaluate the recombinant protein productivity of the bioreactor due to the generation interval from G0 founders to G1 egg-laying hens, to verification of the exogenous protein at the early stage is difficult. Here we propose a system for sequential validation of exogenous protein production in chicken bioreactors as in hatching female chicks as well as in egg-laying hens. We generated chicken OVALBUMIN (OVA) EGFP knock-in (KI) chicken (OVA EGFP KI) by CRISPR/Cas9-mediated nonhomologous end joining at the chicken OVA gene locus. Subsequently, the estrogen analog, diethylstilbestrol (DES), was subcutaneously implanted in the abdominal region of 1-wk-old OVA EGFP KI female chicks to artificially increase OVALBUMIN expression. The oviducts of DES-treated OVA EGFP KI female chicks expressed OVA and EGFP at the 3-wk-old stage (10 d after DES treatment). We evaluated the expression of EGFP protein in the oviduct, along with the physical properties of eggs and egg white from OVA EGFP KI hens. The rapid identification and isolation of exogenous protein can be confirmed at a very early stage and high-yield production is possible by targeting the chicken oviduct.

The distribution of neuron-specific gene family member 1 in brain and germ cells: Implications for the regulation of germ-line development by brain.

Vesicular acidification at early endosomes dissociates endocytosed receptor-ligand complexes. The ligands, receptors, or both are then directed to late endosomes for degradation or recycled back to the plasma membrane. Of neuron-specific gene (NSG) family members, early endosomal protein neuron-specific gene family member 1 (NSG1) is the most important in receptor recycling. In this study, we characterized chicken NSG1 (cNSG1). We found several functional sites related to endocytotic machinery in cNSG1 that were highly conserved with most other vertebrate NSG1 proteins. We examined the tissue and duration specificity and the temporal and spatial patterns of cNSG1 expression. cNSG1 expression was preferentially located in all regions of the brain, neuroendocrine glands, and spinal cord. Unexpectedly, cNSG1 expression was strongly detected during male and female germ-line development. Expression of NSG1 in two apparently unrelated cell types such as neurons and germ cells suggests NSG1 roles in neurons and germ-cells chemotaxis and endocytotic machinery.

In-depth examination of PrPSc in Holstein cattle carrying the E211K somatic mutation of the bovine prion protein gene (PRNP).

Prion diseases are transmissible spongiform encephalopathies caused by deleterious prion protein (PrPSc ) derived from normal prion protein (PrPC ), which is encoded by the prion protein gene (PRNP). We performed an in-depth examination to detect PrPSc by using enzyme immunoassay (EIA), real-time quaking-induced conversion reactions (RT-QuIC) and protein misfolding cyclic amplification (PMCA) in nine brain tissues derived from three Holstein cattle carrying the E211K somatic mutation of the bovine PRNP gene. The EIA, RT-QuIC and PMCA analyses were not able to detect the PrPSc band in any tested samples. To the best of our knowledge, this report is the first to describe an in-depth examination of PrPSc in cattle carrying the E211K somatic mutation of the bovine PRNP gene.

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.

Development and characterization of a CRISPR/Cas9-mediated RAG1 knockout chicken model lacking mature B and T cells.

Although birds have been used historically as a model animal for immunological research, resulting in remarkable achievements, immune cell development in birds themselves has yet to be fully elucidated. In this study, we firstly generated an immunodeficient chicken model using a CRISPR/Cas9-mediated recombination activating gene 1 (RAG1) knockout, to investigate avian-specific immune cell development. Unlike previously reported immunoglobulin (Ig) heavy chain knockout chickens, the proportion and development of B cells in both RAG1 +/- and RAG1 -/- embryos were significantly impaired during B cell proliferation (embryonic day 16 to 18). Our findings indicate that, this is likely due to disordered B cell receptor (BCR)-mediated signaling and interaction of CXC motif chemokine receptor (CXCR4) with CXCL12, resulting from disrupted Ig V(D)J recombination at the embryonic stage. Histological analysis after hatching showed that, unlike wild-type (WT) and RAG1 +/- chickens, lymphatic organs in 3-week old RAG1 -/- chickens were severely damaged. Furthermore, relative to WT chickens, RAG1+/- and RAG1-/- birds had reduced serum Igs, fewer mature CD4+ and CD8+ T lymphocytes. Furthermore, BCR-mediated B cell activation in RAG1 +/- chickens was insufficient, leading to decreased expression of the activation-induced deaminase (AID) gene, which is important for Ig gene conversion. Overall, this immunodeficient chicken model underlines the pivotal role of RAG1 in immature B cell development, Ig gene conversion during embryonic stages, and demonstrates the dose-dependent regulatory role of RAG1 during immune cell development. This model will provide ongoing insights for understanding chicken immune system development and applied in the fields of immunology and biomedical science.

Efficient production of recombinant human adiponectin in egg white using genome edited chickens.

The prevalence of obesity-related metabolic diseases caused by insulin resistance is rapidly increasing worldwide. Adiponectin (ADPN), a hormone derived from adipose tissue, is a potential therapeutic agent for insulin resistance. Chickens are considered efficient bioreactors for recombinant protein production because they secrete large amounts of high-concentration proteins from the oviduct. Additionally, chickens express high levels of high-molecular-weight (HMW) ADPN, which is considered the active form in the body. Therefore, in this study, a gene-targeted chicken model was produced in which the gene encoding human ADPN was inserted into Ovalbumin (OVA) using the CRISPR/Cas9 system, and the characteristics of the resulting recombinant ADPN protein were evaluated. As a result, human ADPN was expressed in G1 hen oviducts and egg whites of OVA ADPN knock-in (KI) chickens. The concentration of ADPN in egg white ranged from 1.47 to 4.59 mg/mL, of which HMW ADPN accounted for ∼29% (0.24-1.49 mg/mL). Importantly, egg white-derived ADPN promoted expression of genes related to fatty acid oxidation and activated the 5'-AMP-activated protein kinase (AMPK) signaling pathway in muscle cells. In summary, the OVA gene-targeted chicken bioreactor proved to be an advantageous model for production of human ADPN, and the resulting protein was of sufficient quantity and efficacy for industrial use.

First report of a strong association between genetic polymorphisms of the prion protein gene (PRNP) and susceptibility to chronic wasting disease in sika deer (Cervus nippon).

Prion diseases are incurable neurodegenerative disorders caused by proteinase K-resistant prion protein (PrPSc ) derived from normal prion protein (PrPC ) encoded by the prion protein gene (PRNP). Although the cervid PRNP gene plays a pivotal role in the pathological mechanism of chronic wasting disease (CWD), there is no existing association analysis between susceptibility to CWD and genetic polymorphisms of the PRNP gene in sika deer. We investigated genetic polymorphisms of the PRNP gene using amplicon sequencing in sika deer. In addition, to identify a genetic susceptibility factor, we compared the genotype, allele and haplotype frequencies of the PRNP gene between CWD-positive and CWD-negative sika deer. Furthermore, to assess the effect of the genetic polymorphisms on sika deer prion protein (PrP), we performed in silico analysis using PolyPhen-2, PROVEAN and AMYCO. Finally, we analysed the tertiary structure and electrostatic potential of sika deer PrP based on single nucleotide polymorphisms (SNPs) using the SWISS-MODEL and Swiss-PdbViewer programs. We found a total of 24 SNPs of the PRNP gene, including 22 novel SNPs (10 synonymous SNPs and 12 nonsynonymous SNPs), in sika deer. Among the nonsynonymous SNPs, we found a strong association of susceptibility to CWD with c.56G > A (Ser19Asn). In addition, we found that c.56G > A (Ser19Asn), c.296A > T (His99Leu) and c.560T > A (Val187Asp) were predicted to have damaging effects on sika deer PrP. Furthermore, we observed significant alterations in the electrostatic potential of sika deer PrP by genetic polymorphisms of the 187Asp allele. To the best of our knowledge, this was the first association study between genetic polymorphisms of the PRNP gene and susceptibility to CWD in sika deer.

Dissecting chicken germ cell dynamics by combining a germ cell tracing transgenic chicken model with single-cell RNA sequencing.

Avian germ cells can be distinguished by certain characteristics during development. On the basis of these characteristics, germ cells can be used for germline transmission. However, the dynamic transcriptional landscape of avian germ cells during development is unknown. Here, we used a novel germ-cell-tracing method to monitor and isolate chicken germ cells at different stages of development. We targeted the deleted in azoospermia like (DAZL) gene, a germ-cell-specific marker, to integrate a green fluorescent protein (GFP) reporter gene without affecting endogenous DAZL expression. The resulting transgenic chickens (DAZL::GFP) were used to uncover the dynamic transcriptional landscape of avian germ cells. Single-cell RNA sequencing of 4,752 male and 13,028 female DAZL::GFP germ cells isolated from embryonic day E2.5 to 1 week post-hatch identified sex-specific developmental stages (4 stages in male and 5 stages in female) and trajectories (apoptosis and meiosis paths in female) of chicken germ cells. The male and female trajectories were characterized by a gradual acquisition of stage-specific transcription factor activities. We also identified evolutionary conserved and species-specific gene expression programs during both chicken and human germ-cell development. Collectively, these novel analyses provide mechanistic insights into chicken germ-cell development.

In vivo enrichment of busulfan-resistant germ cells for efficient production of transgenic avian models.

Most transgenic animals are generated using a genome-modified stem cell system and genome modification directly in embryos. Although this system is well-established in the development of transgenic animals, donor cell-derived transgenic animal production is inefficient in some cases. Especially in avian models such as chickens, the efficiency of transgenic animal production through primordial germ cells (PGCs) is highly variable compared with embryonic manipulation of mammalian species. Because germ cell and germline-competent stem cell-mediated systems that contain the transgene are enriched only at the upstream level during cell cultivation, the efficiency of transgenic animal production is unreliable. Therefore, we developed an in vivo selection model to enhance the efficiency of transgenic chicken production using microsomal glutathione-S-transferase II (MGSTII)-overexpressing PGCs that are resistant to the alkylating agent busulfan, which induces germ cell-specific cytotoxicity. Under in vitro conditions, MGSTII-tg PGCs were resistant to 1 µM busulfan, which was highly toxic to wild-type PGCs. In germline chimeric roosters, transgene-expressing germ cells were dominantly colonized in the recipient testes after busulfan exposure compared with non-treated germline chimera. In validation of germline transmission, donor PGC-derived progeny production efficiency was 94.68%, and the transgene production rate of heterozygous transgenic chickens was significantly increased in chickens that received 40 mg/kg busulfan (80.33-95.23%) compared with that of non-treated germline chimeras (51.18%). This system is expected to significantly improve the efficiency of generating transgenic chickens and other animal species by increasing the distribution of donor cells in adult testes.

Association Study of the M132L Single Nucleotide Polymorphism With Susceptibility to Chronic Wasting Disease in Korean Elk: A Meta-Analysis.

Chronic wasting disease (CWD) is a deleterious brain proteinopathy caused by a pathogenic form of prion protein (PrPSc), which is converted from a benign form of prion protein (PrPC) encoded by the prion protein gene (PRNP). In elk, the M132L single nucleotide polymorphism (SNP) of the PRNP gene likely plays a pivotal role in susceptibility to CWD. However, the association of the M132L SNP with susceptibility to CWD has not been evaluated in Korean elk to date. To estimate the association of the M132L SNP with susceptibility to CWD in Korean elk, we investigated the genotype and allele frequencies of the M132L SNP by amplicon sequencing and performed association analysis between CWD-positive and CWD-negative elk. In addition, we performed a meta-analysis to evaluate the association between the M132L SNP and susceptibility to CWD in quantitatively synthesized elk populations. Furthermore, we estimated the effect of the M132L SNP on elk PrP using in silico programs, including PolyPhen-2, PROVEAN, AMYCO and Swiss-PdbViewer. We did not identify a significant association between the M132L SNP of PRNP and susceptibility to CWD in Korean elk. The meta-analysis also did not identify a strong association between the M132L SNP of PRNP and susceptibility to CWD in quantitatively synthesized elk populations. Furthermore, we did not observe significant changes in structure, amyloid propensity or electrostatic potential based on the M132L SNP in elk PrP. To the best of our knowledge, this was the first report of an association analysis and meta-analysis in Korean elk and quantitatively synthesized elk populations, respectively.

Migration and proliferation of intact and genetically modified primordial germ cells and the generation of a transgenic chicken.

This study evaluated gonadal migration and postmigratory proliferation of intact and genetically modified chicken primordial germ cells (PGCs). A randomized, controlled trial was conducted with the gonadal population of PGCs and transgenic chicken production as major parameters. PGCs (0, 90, 900, 1800, or 3000 cells) were transferred into 53-h-old embryos. The percentage of PGCs migrating on Day 6 of development was highest (35.8%) following the transfer of 900 PGCs and did not change with increases in transferred PGCs. The number of migrating PGCs gradually increased (P = 0.0001) as the number of transferred PGCs was increased. Gonadal migration was detected after the transfer of intact and genetically modified PGCs, but prominent decreases in PGC migration (from 21.9% to 0.38%) and chimera ratio (from 0.4 to 0.007) occurred with genetically modified PGCs. However, subsequent vigorous proliferation of the modified PGCs (3.67-fold increase from transferred number) led to the derivation of a germline chimera and produced a transgenic hatchling. In conclusion, the number of migrating PGCs increased as the number of transferred cells increased. Vigorous proliferation after transfer compensated for the decreased migration capacity of genetically modified PGCs and resulted in the production of a transgenic chicken.

Production of germline chimeric quails by transplantation of cryopreserved testicular cells into developing embryos.

The germplasm is a resource and tool for the conservation of genetic diversity in animals, including birds. Securing germplasm is limited in most bird species due to difficulties in semen collection and germ cell isolation, lack of germ cell-specific markers, and in vitro culture systems. Here, we report the production of germline chimeric quails by transplant of cryopreserved testicular cells (TCs) into the developing embryo. The testicular germ cell properties were maintained after freeze-thaw, with no significant reduction in cell viability irrespective of storage length. Cryopreserved TCs were transferred into Hamburger Hamilton (HH) stage 14-17 quail embryos, and were demonstrated to migrate into the embryonic gonads with similar efficiency to freshly isolated TCs. Twenty of 81 recipient embryos yielded hatchlings from cryopreserved TCs and the germline transmission efficiency was similar to that of freshly isolated cells. In conclusion, cryopreserved adult quail TCs are capable of (de)differentiation into functional gametes in recipient quail gonads and can generate donor TCs-derived progenies. This system is feasible for the isolation of sufficient germplasm resources from various bird species for conservation purposes.