Abstract: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 sol… Show more
“…6 b) and viability between 90 and 95% (Table S5 ). Thus the knock-out PGC clones had comparable characteristics as the germline colonizing PGC described before 39 . Figure 6 Committed stem cell character and growth curves of PGCs.…”
Section: Resultsmentioning
confidence: 63%
“…Lohmann Breeders GmbH (Cuxhaven, Germany) donated fertilized eggs from an intercross of a population that was heterozygous for the blue eggshell color locus. For our experiments, we used a homozygous blue-allele bearing cell line which was established from this intercross in previous work 39 . These eggs derived from a case study which was part of the EU-project Innovative Management of Animal Genetic resources (IMAGE).…”
Section: Methodsmentioning
confidence: 99%
“…Reaction products were resolved using a 1.5% ultrapure agarose (Invitrogen) gel electrophoresis run at 80 V for 45 min in 1× TBE-buffer, and visualized using a transilluminator. Intron-spanning primer were used as in our previous studies 39 .…”
Primordial germ cells (PGCs), the precursors of sperm and oocytes, pass on the genetic material to the next generation. The previously established culture system of chicken PGCs holds many possibilities for functional genomics studies and the rapid introduction of desired traits. Here, we established a CRISPR/Cas9-mediated genome editing protocol for the genetic modification of PGCs derived from chickens with blue eggshell color. The sequence targeted in the present report is a provirus (EAV-HP) insertion in the 5’-flanking region of the SLCO1B3 gene on chromosome 1 in Araucana chickens, which is supposedly responsible for the blue eggshell color. We designed pairs of guide RNAs (gRNAs) targeting the entire 4.2 kb provirus region. Following transfection of PGCs with the gRNA, genomic DNA was isolated and analyzed by mismatch cleavage assay (T7EI). For absolute quantification of the targeting efficiencies in homozygous blue-allele bearing PGCs a digital PCR was established, which revealed deletion efficiencies of 29% when the wildtype Cas9 was used, and 69% when a high-fidelity Cas9 variant was employed. Subsequent single cell dilutions of edited PGCs yielded 14 cell clones with homozygous deletion of the provirus. A digital PCR assay proved the complete absence of this provirus in cell clones. Thus, we demonstrated the high efficiency of the CRISPR/Cas9 system in introducing a large provirus deletion in chicken PGCs. Our presented workflow is a cost-effective and rapid solution for screening the editing success in transfected PGCs.
“…6 b) and viability between 90 and 95% (Table S5 ). Thus the knock-out PGC clones had comparable characteristics as the germline colonizing PGC described before 39 . Figure 6 Committed stem cell character and growth curves of PGCs.…”
Section: Resultsmentioning
confidence: 63%
“…Lohmann Breeders GmbH (Cuxhaven, Germany) donated fertilized eggs from an intercross of a population that was heterozygous for the blue eggshell color locus. For our experiments, we used a homozygous blue-allele bearing cell line which was established from this intercross in previous work 39 . These eggs derived from a case study which was part of the EU-project Innovative Management of Animal Genetic resources (IMAGE).…”
Section: Methodsmentioning
confidence: 99%
“…Reaction products were resolved using a 1.5% ultrapure agarose (Invitrogen) gel electrophoresis run at 80 V for 45 min in 1× TBE-buffer, and visualized using a transilluminator. Intron-spanning primer were used as in our previous studies 39 .…”
Primordial germ cells (PGCs), the precursors of sperm and oocytes, pass on the genetic material to the next generation. The previously established culture system of chicken PGCs holds many possibilities for functional genomics studies and the rapid introduction of desired traits. Here, we established a CRISPR/Cas9-mediated genome editing protocol for the genetic modification of PGCs derived from chickens with blue eggshell color. The sequence targeted in the present report is a provirus (EAV-HP) insertion in the 5’-flanking region of the SLCO1B3 gene on chromosome 1 in Araucana chickens, which is supposedly responsible for the blue eggshell color. We designed pairs of guide RNAs (gRNAs) targeting the entire 4.2 kb provirus region. Following transfection of PGCs with the gRNA, genomic DNA was isolated and analyzed by mismatch cleavage assay (T7EI). For absolute quantification of the targeting efficiencies in homozygous blue-allele bearing PGCs a digital PCR was established, which revealed deletion efficiencies of 29% when the wildtype Cas9 was used, and 69% when a high-fidelity Cas9 variant was employed. Subsequent single cell dilutions of edited PGCs yielded 14 cell clones with homozygous deletion of the provirus. A digital PCR assay proved the complete absence of this provirus in cell clones. Thus, we demonstrated the high efficiency of the CRISPR/Cas9 system in introducing a large provirus deletion in chicken PGCs. Our presented workflow is a cost-effective and rapid solution for screening the editing success in transfected PGCs.
“…PGCs were derived from chicken embryonic gonads (E7, stage HH 28–29) and cultured as described 49 . The PGCs were cultured in suspension without a feeder-layer in customized avian KO-DMEM (23).…”
Bucky ball was identified as germ plasm organizer in zebrafish and has proven crucial for Balbiani body condensation. A synteny comparison identified an uncharacterized gene locus in the chicken genome as predicted avian counterpart. Here, we present experimental evidence that this gene locus indeed encodes a ‘Bucky ball’ equivalent in matured oocytes and early embryos of chicken. Heterologous expression of Bucky ball fusion proteins both from zebrafish and chicken with a fluorescent reporter revealed unique patterns indicative for liquid–liquid phase separation of intrinsically disordered proteins. Immuno-labeling detected Bucky ball from oocytes to blastoderms with diffuse distribution in matured oocytes, aggregation in first cleavage furrows, and co-localization to the chicken vasa homolog (CVH). Later, Bucky ball translocated to the cytoplasm of first established cells, and showed nuclear translocation during the major zygotic activation together with CVH. Remarkably, during the phase of area pellucida formation, Bucky ball translocated back into the cytoplasm at stage EGK VI, whereas CVH remained within the nuclei. The condensation of Bucky ball and co-localization with CVH in cleavage furrows and nuclei of the centrally located cells strongly suggests chicken Bucky ball as a germ plasm organizer in birds, and indicate a special importance of the major zygotic activation for germline specification.
“…Alternatively, completely removing embryos from their natural eggshells to various types of recently developed artificial vessels for further observation of developmental changes and genetic manipulation is called ex ovo studies. To provide in-depth studies into avian embryogenesis, parts of embryos were taken to derive cell lines in in vitro culture including embryonic stem cells (ESCs) and primordial germ cells (PGCs), and become an essential tool to understand pluripotency network regulating early development ( van de Lavoir et al, 2006 ; Choi et al, 2010 ; Aubel and Pain, 2013 ; Whyte et al, 2015 ; Altgilbers et al, 2021 ). In vitro culture with genetic manipulation also enables us to study the gain/loss of functions and perform cellular reprogramming to produce induced pluripotent stem cells (iPSCs) capable of differentiation into all cell types, similar to ESCs ( Dai et al, 2014 ; Katayama et al, 2018 ).…”
The avian embryos growing outside the natural eggshell (ex ovo) were observed since the early 19th century, and since then chick embryonic structures have revealed reaching an in-depth view of external and internal anatomy, enabling us to understand conserved vertebrate development. However, the internal environment within an eggshell (in ovo) would still be the ideal place to perform various experiments to understand the nature of avian development and to apply other biotechnology techniques. With the advent of genetic manipulation and cell culture techniques, avian embryonic parts were dissected for explant culture to eventually generate expandable cell lines (in vitro cell culture). The expansion of embryonic cells allowed us to unravel the transcriptional network for understanding pluripotency and differentiation mechanism in the embryos and in combination with stem cell technology facilitated the applications of avian culture to the next levels in transgenesis and wildlife conservation. In this review, we provide a panoramic view of the relationship among different cultivation platforms from in ovo studies to ex ovo as well as in vitro culture of cell lines with recent advances in the stem cell fields.
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