Loss of αß but not γδ T cells in chickens causes a severe phenotype.

The availability of genetically modified mice has facilitated the study of mammalian T cells. No model has yet been developed to study these cells in chickens, an important livestock species with a high availability of γδ T cells. To investigate the role of γδ and αß T cell populations in birds, we generated chickens lacking these T cell populations. This was achieved by genomic deletion of the constant region of the T cell receptor γ or ß chain, leading to a complete loss of either γδ or αß T cells. Our results show that a deletion of αß T cells but not γδ T cells resulted in a severe phenotype in KO chickens. The αß T cell KO chickens exhibited granulomas associated with inflammation of the spleen and the proventriculus. Immunophenotyping of αß T cell KO chickens revealed a significant increase in monocytes and expectedly the absence of CD4+ T cells including FoxP3+ regulatory T cells. Surprisingly there was no increase of γδ T cells. In addition, we observed a significant decrease in immunoglobulins, B lymphocytes, and changes in the bursa morphology. Our data reveal the consequences of T cell knockouts in chickens and provide new insights into their function in vertebrates.

AP-2δ Expression Kinetics in Multimodal Networks in the Developing Chicken Midbrain.

AP-2 is a family of transcription factors involved in many aspects of development, cell differentiation, and regulation of cell growth and death. AP-2δ is a member of this group and specific gene expression patterns are required in the adult mouse brain for the development of parts of the inferior colliculus (IC), as well as the cortex, dorsal thalamus, and superior colliculus. The midbrain is one of the central areas in the brain where multimodal integration, i.e., integration of information from different senses, occurs. Previous data showed that AP-2δ-deficient mice are viable but due to increased apoptosis at the end of embryogenesis, lack part of the posterior midbrain. Despite the absence of the IC in AP-2δ-deficient mice, these animals retain at least some higher auditory functions. Neuronal responses to tones in the neocortex suggest an alternative auditory pathway that bypasses the IC. While sufficient data are available in mammals, little is known about AP-2δ in chickens, an avian model for the localization of sounds and the development of auditory circuits in the brain. Here, we identified and localized AP-2δ expression in the chicken midbrain during embryogenesis. Our data confirmed the presence of AP-2δ in the inferior colliculus and optic tectum (TeO), specifically in shepherd's crook neurons, which are an essential component of the midbrain isthmic network and involved in multimodal integration. AP-2δ expression in the chicken midbrain may be related to the integration of both auditory and visual afferents in these neurons. In the future, these insights may allow for a more detailed study of circuitry and computational rules of auditory and multimodal networks.

A Genetically Engineered Commercial Chicken Line Is Resistant to Highly Pathogenic Avian Leukosis Virus Subgroup J.

Viral diseases remain a major concern for animal health and global food production in modern agriculture. In chickens, avian leukosis virus subgroup J (ALV-J) represents an important pathogen that causes severe economic loss. Until now, no vaccine or antiviral drugs are available against ALV-J and strategies to combat this pathogen in commercial flocks are desperately needed. CRISPR/Cas9 targeted genome editing recently facilitated the generation of genetically modified chickens with a mutation of the chicken ALV-J receptor Na+/H+ exchanger type 1 (chNHE1). In this study, we provide evidence that this mutation protects a commercial chicken line (NHE1ΔW38) against the virulent ALV-J prototype strain HPRS-103. We demonstrate that replication of HPRS-103 is severely impaired in NHE1ΔW38 birds and that ALV-J-specific antigen is not detected in cloacal swabs at later time points. Consistently, infected NHE1ΔW38 chickens gained more weight compared to their non-transgenic counterparts (NHE1W38). Histopathology revealed that NHE1W38 chickens developed ALV-J typical pathology in various organs, while no pathological lesions were detected in NHE1ΔW38 chickens. Taken together, our data revealed that this mutation can render a commercial chicken line resistant to highly pathogenic ALV-J infection, which could aid in fighting this pathogen and improve animal health in the field.

Cas9-expressing chickens and pigs as resources for genome editing in livestock.

Genetically modified animals continue to provide important insights into the molecular basis of health and disease. Research has focused mostly on genetically modified mice, although other species like pigs resemble the human physiology more closely. In addition, cross-species comparisons with phylogenetically distant species such as chickens provide powerful insights into fundamental biological and biomedical processes. One of the most versatile genetic methods applicable across species is CRISPR-Cas9. Here, we report the generation of transgenic chickens and pigs that constitutively express Cas9 in all organs. These animals are healthy and fertile. Functionality of Cas9 was confirmed in both species for a number of different target genes, for a variety of cell types and in vivo by targeted gene disruption in lymphocytes and the developing brain, and by precise excision of a 12.7-kb DNA fragment in the heart. The Cas9 transgenic animals will provide a powerful resource for in vivo genome editing for both agricultural and translational biomedical research, and will facilitate reverse genetics as well as cross-species comparisons.