Chickens with a Truncated Light Chain Transgene Express Single-Domain H Chain-Only Antibodies.

H chain-only Igs are naturally produced in camelids and sharks. Because these Abs lack the L chain, the Ag-binding domain is half the size of a traditional Ab, allowing this type of Ig to bind to targets in novel ways. Consequently, the H chain-only single-domain Ab (sdAb) structure has the potential to increase the repertoire and functional range of an active humoral immune system. The majority of vertebrates use the standard heterodimeric (both H and L chains) structure and do not produce sdAb format Igs. To investigate if other animals are able to support sdAb development and function, transgenic chickens (Gallus gallus) were designed to produce H chain-only Abs by omitting the L chain V region and maintaining only the LC region to serve as a chaperone for Ab secretion from the cell. These birds produced 30-50% normal B cell populations within PBMCs and readily expressed chicken sequence sdAbs. Interestingly, the H chains contained a spontaneous CH1 deletion. Although no isotype switching to IgY or IgA occurred, the IgM repertoire was diverse, and immunization with a variety of protein immunogens rapidly produced high and specific serum titers. mAbs of high affinity were efficiently recovered by single B cell screening. In in vitro functional assays, the sdAbs produced by birds immunized against SARS-CoV-2 were also able to strongly neutralize and prevent viral replication. These data suggest that the truncated L chain design successfully supported sdAb development and expression in chickens.

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.

Chickens with humanized immunoglobulin genes generate antibodies with high affinity and broad epitope coverage to conserved targets.

Transgenic animal platforms for the discovery of human monoclonal antibodies have been developed in mice, rats, rabbits and cows. The immune response to human proteins is limited in these animals by their tolerance to mammalian-conserved epitopes. To expand the range of epitopes that are accessible, we have chosen an animal host that is less phylogenetically related to humans. Specifically, we generated transgenic chickens expressing antibodies from immunoglobulin heavy and light chain loci containing human variable regions and chicken constant regions. From these birds, paired human light and heavy chain variable regions are recovered and cloned as fully human recombinant antibodies. The human antibody-expressing chickens exhibit normal B cell development and raise immune responses to conserved human proteins that are not immunogenic in mice. Fully human monoclonal antibodies can be recovered with sub-nanomolar affinities. Binning data of antibodies to a human protein show epitope coverage similar to wild type chickens, which we previously showed is broader than that produced from rodent immunizations.

Expression of heavy chain-only antibodies can support B-cell development in light chain knockout chickens.

Since the discovery of antibody-producing B cells in chickens six decades ago, chickens have been a model for B-cell development in gut-associated lymphoid tissue species. Here we describe targeting of the immunoglobulin light chain locus by homologous recombination in chicken primordial germ cells (PGCs) and generation of VJCL knockout chickens. In contrast to immunoglobulin heavy chain knockout chickens, which completely lack mature B cells, homozygous light chain knockout (IgL(-/-) ) chickens have a small population of B lineage cells that develop in the bursa and migrate to the periphery. This population of B cells expresses the immunoglobulin heavy chain molecule on the cell surface. Soluble heavy-chain-only IgM and IgY proteins of reduced molecular weight were detectable in plasma in 4-week-old IgL(-/-) chickens, and antigen-specific IgM and IgY heavy chain proteins were produced in response to immunization. Circulating heavy-chain-only IgM showed a deletion of the CH1 domain of the constant region enabling the immunoglobulin heavy chain to be secreted in the absence of the light chain. Our data suggest that the heavy chain by itself is enough to support all the important steps in B-cell development in a gut-associated lymphoid tissue species.

Generation of chickens expressing Cre recombinase.

Cre recombinase has been extensively used for genome engineering in transgenic mice yet its use in other species has been more limited. Here we describe the generation of transgenic chickens expressing Cre recombinase. Green fluorescent protein (GFP)-positive chicken primordial germ cells were stably transfected with ß-actin-Cre-recombinase using phiC31 integrase and transgenic chickens were generated. Cre recombinase activity was verified by mating Cre birds to birds carrying a floxed transgene. Floxed sequences were only excised in offspring from roosters that inherited the Cre recombinase but were excised in all offspring from hens carrying the Cre recombinase irrespective of the presence of the Cre transgene. The Cre recombinase transgenic birds were healthy and reproductively normal. The Cre and GFP genes in two of the lines were closely linked whereas the genes segregated independently in a third line. These founders allowed development of GFP-expressing and non-GFP-expressing Cre recombinase lines. These lines of birds create a myriad of opportunities to study developmentally-regulated and tissue-specific expression of transgenes in chickens.

Germline Gene Editing in Chickens by Efficient CRISPR-Mediated Homologous Recombination in Primordial Germ Cells.

The CRISPR/Cas9 system has been applied in a large number of animal and plant species for genome editing. In chickens, CRISPR has been used to knockout genes in somatic tissues, but no CRISPR-mediated germline modification has yet been reported. Here we use CRISPR to target the chicken immunoglobulin heavy chain locus in primordial germ cells (PGCs) to produce transgenic progeny. Guide RNAs were co-transfected with a donor vector for homology-directed repair of the double-strand break, and clonal populations were selected. All of the resulting drug-resistant clones contained the correct targeting event. The targeted cells gave rise to healthy progeny containing the CRISPR-targeted locus. The results show that gene-edited chickens can be obtained by modifying PGCs in vitro with the CRISPR/Cas9 system, opening up many potential applications for efficient genetic modification in birds.

Inserting random and site-specific changes into the genome of chickens.

During the past decade, modifications to the chicken genome have evolved from random insertions of small transgenes using viral vectors to site-specific deletions using homologous recombination vectors and nontargeted insertions of large transgenes using phi-31 integrase. Primordial germ cells (PGC) and gonocytes are the germline-competent cell lines in which targeted modifications and large transgenes are inserted into the genome. After extended periods of in vitro culture, PGC retain their capacity to form functional gametes when reintroduced in vivo. Rates of stable germline modification vary from 1×10(-5) for nontargeted insertions to 1×10(-8) for targeted insertions. Following transfection, clonally derived cell lines are expanded, injected into Stage 13-15 Hamburger and Hamilton embryos, and putative chimeras are incubated to term in surrogate shells. Green fluorescent protein (GFP) is incorporated into transgenes to reveal the presence of genetically modified PGC in culture and the extent of colonization of the gonad during the first week posthatch. If the extent of colonization is adequate, cohorts of putative chimeras are reared to sexual maturity. Semen is collected and the contribution from donor PGC is estimated by evaluating GFP expression using flow cytometry and PCR. The most promising candidates are selected for breeding to obtain G1 heterozygote offspring. To date, this protocol has been used to (1) knockout the immunoglobulin heavy and light chain genes and produce chickens lacking humoral immunity, (2) insert human V genes and arrays of pseudo V genes into the heavy and light immunoglobulin loci to produce chickens making antibodies with human V regions, (3) insert GFP into nontargeted locations within the genome to produce chickens expressing GFP, and (4) insert Cre recombinase into the genome to produce chickens that excise sequences of DNA flanked by loxP sites.

Immunoglobulin knockout chickens via efficient homologous recombination in primordial germ cells.

Gene targeting by homologous recombination or by sequence-specific nucleases allows the precise modification of genomes and genes to elucidate their functions. Although gene targeting has been used extensively to modify the genomes of mammals, fish, and amphibians, a targeting technology has not been available for the avian genome. Many of the principles of humoral immunity were discovered in chickens, yet the lack of gene targeting technologies in birds has limited biomedical research using this species. Here we describe targeting the joining (J) gene segment of the chicken Ig heavy chain gene by homologous recombination in primordial germ cells to establish fully transgenic chickens carrying the knockout. In homozygous knockouts, Ig heavy chain production is eliminated, and no antibody response is elicited on immunization. Migration of B-lineage precursors into the bursa of Fabricius is unaffected, whereas development into mature B cells and migration from the bursa are blocked in the mutants. Other cell types in the immune system appear normal. Chickens lacking the peripheral B-cell population will provide a unique experimental model to study avian immune responses to infectious disease. More generally, gene targeting in avian primordial germ cells will foster advances in diverse fields of biomedical research such as virology, stem cells, and developmental biology, and provide unique approaches in biotechnology, particularly in the field of antibody discovery.

Interspecific germline transmission of cultured primordial germ cells.

In birds, the primordial germ cell (PGC) lineage separates from the soma within 24 h following fertilization. Here we show that the endogenous population of about 200 PGCs from a single chicken embryo can be expanded one million fold in culture. When cultured PGCs are injected into a xenogeneic embryo at an equivalent stage of development, they colonize the testis. At sexual maturity, these donor PGCs undergo spermatogenesis in the xenogeneic host and become functional sperm. Insemination of semen from the xenogeneic host into females from the donor species produces normal offspring from the donor species. In our model system, the donor species is chicken (Gallus domesticus) and the recipient species is guinea fowl (Numida meleagris), a member of a different avian family, suggesting that the mechanisms controlling proliferation of the germline are highly conserved within birds. From a pragmatic perspective, these data are the basis of a novel strategy to produce endangered species of birds using domesticated hosts that are both tractable and fecund.

Genetic modification of primordial germ cells by gene trapping, gene targeting, and phiC31 integrase.

The genome of germline committed cells is thought to be protected by mechanisms of transcriptional silencing, posing a barrier to transgenesis using cultured germline cells. We found that selection for transgene integration into the primordial germ cell genome required that the transgenes be flanked by the chicken beta-globin insulator. However, integration frequency was low, and sequencing of the insertion sites revealed that the transgenes preferentially inserted into active promoter regions, implying that silencing prohibited recovery of insertions in other regions. Much higher frequencies of integration were achieved when the phiC31 integrase was used to insert transgenes into endogenous pseudo attP sites. Despite the evidence for transcriptional silencing in PGCs, gene targeting of a nonexpressed gene was also achieved. The ability to make genetic modifications in PGCs provides unprecedented opportunities to study the biology of PGCs, as well as produce transgenic chickens for applications in biotechnology and developmental biology.

Avian embryonic stem cells.

Blastodermal cells derived from the area pellucida of a stage X (EG&K) embryo have the potential to contribute to the somatic tissues and the germ line when reintroduced into a stage X (EG&K) recipient embryo. This chapter describes a method to culture chicken embryonic stem (cES) cells derived from blastodermal cells. Within the first week of culture, the cells change their morphology; they become smaller with a large nucleus and a prominent nucleolus. The cES cells remain chromosomally normal and can be cultured for extended periods. They can be modified genetically using standard electroporation procedures and, after injection into a recipient embryo, can contribute to all somatic tissues. Using a surrogate shell culture system, the injected embryos can be manipulated and visualized easily throughout incubation. We have generated high-grade chimeras by compromising the recipient embryos and maintaining the ES cells in stage X (EG&K) recipients for a few days at 15 degrees before incubating them at 37.5 degrees. The cES system provides a novel experimental paradigm for the investigation of developmental and physiological mechanisms in the chicken.

Germline transmission of genetically modified primordial germ cells.

Primordial germ cells (PGCs) are the precursors of sperm and eggs. In most animals, segregation of the germ line from the somatic lineages is one of the earliest events in development; in avian embryos, PGCs are first identified in an extra-embryonic region, the germinal crescent, after approximately 18 h of incubation. After 50-55 h of development, PGCs migrate to the gonad and subsequently produce functional sperm and oocytes. So far, cultures of PGCs that remain restricted to the germ line have not been reported in any species. Here we show that chicken PGCs can be isolated, cultured and genetically modified while maintaining their commitment to the germ line. Furthermore, we show that chicken PGCs can be induced in vitro to differentiate into embryonic germ cells that contribute to somatic tissues. Retention of the commitment of PGCs to the germ line after extended periods in culture and after genetic modification combined with their capacity to acquire somatic competence in vitro provides a new model for developmental biology. The utility of the model is enhanced by the accessibility of the avian embryo, which facilitates access to the earliest stages of development and supplies a facile route for the reintroduction of PGCs into the embryonic vasculature. In addition, these attributes create new opportunities to manipulate the genome of chickens for agricultural and pharmaceutical applications.

High-grade transgenic somatic chimeras from chicken embryonic stem cells.

Male and female embryonic stem (ES) cell lines were derived from the area pellucidae of Stage X (EG&K) chicken embryos. These ES cell lines were grown in culture for extended periods of time and the majority of the cells retained a diploid karyotype. When reintroduced into Stage VI-X (EG&K) recipient embryos, the cES cells were able to contribute to all somatic tissues. By combining irradiation of the recipient embryo with exposure of the cES cells to the embryonic environment in diapause, a high frequency and extent of chimerism was obtained. High-grade chimeras, indistinguishable from the donor phenotype by feather pigmentation, were produced. A transgene encoding GFP was incorporated into the genome of cES cells under control of the ubiquitous promoter CX and GFP was widely expressed in somatic tissues. Although cES cells made extensive contributions to the somatic tissues, contribution to the germline was not observed.

Production of human monoclonal antibody in eggs of chimeric chickens.

The tubular gland of the chicken oviduct is an attractive system for protein expression as large quantities of proteins are deposited in the egg, the production of eggs is easily scalable and good manufacturing practices for therapeutics from eggs have been established. Here we examined the ability of upstream and downstream DNA sequences of ovalbumin, a protein produced exclusively in very high quantities in chicken egg white, to drive tissue-specific expression of human mAb in chicken eggs. To accommodate these large regulatory regions, we established and transfected lines of chicken embryonic stem (cES) cells and formed chimeras that express mAb from cES cell-derived tubular gland cells. Eggs from high-grade chimeras contained up to 3 mg of mAb that possesses enhanced antibody-dependent cellular cytotoxicity (ADCC), nonantigenic glycosylation, acceptable half-life, excellent antigen recognition and good rates of internalization.