Robert J. Etches

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.

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.

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.

Sequences in antibody molecules important for receptor-mediated transport into the chicken egg yolk

Large quantities of antibodies are transported into the yolk of the chickens egg. We have identified several regions within the antibody molecule important for its uptake into the egg yolk. An intact Fc and hinge region but not the Fc-associated carbohydrate are required for transport. Our data suggest that the C(H)2/C(H)3 interface is recognized by the receptor responsible for immunoglobulin (Ig) transport. At this interface, residues 251-254 form an exposed loop on the surface of C(H)2. Chicken IgY (cIgY) has the sequence LYIS and human IgG (hIgG) has the sequence LMIS at these positions; mutation of MIS to glycines results in an IgG that is not transported. A second site important for transport is at positions 429-432 within C(H)3. All transported antibodies have the sequence HEAL, whereas, murine IgG2b (mIgG2b) with the sequence HEGL and cIgA with the sequence HDGI fail to be transported. hIgA has the HEAL sequence and is transported.

Immunoglobulin knockout chickens via efficient homologous recombination in primordial germ cells

Significance Chickens have been an important animal model in the fields of developmental biology and immunology over the last century and have contributed a number of basic findings in these areas. However, their use has been limited more recently because there has been no way to genetically engineer specific mutations in the chicken. In addition to basic research applications, genetic modification in birds could also be used to mitigate the threat of avian influenza by production of flu-resistant bird stocks in agriculture. Here we describe an efficient way to target genes of interest in the chicken genome and create knockout chickens. We targeted the immunoglobulin heavy chain gene, leading to loss of antibody production and a block in B-cell development. 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.

Deposition of genetically engineered human antibodies into the egg yolk of hens

To determine if human immunoglobulins (hIg) are capable of being transported into the hens egg, 10 microg each of purified hIgG and hIgA were intravenously injected into SC Hyline(TM) hens and their presence in egg yolk and egg white was determined by ELISA. In both cases deposition into the egg yolk was observed and in the case of hIgA, deposition was also observed in the egg white. Two stably transfected DT40 cell lines secreting recombinant human IgG3 and IgA (rhIgG3 and rhIgA) were injected into laying hens. The DT40 cells colonized the host and rhIgG3 and rhIgA were deposited in egg yolk. Deposition of rhIgA was also observed in the egg white. These data demonstrate that human immunoglobulins and other foreign proteins may be targeted to the chickens egg. In view of the high rate of reproduction, the short generation interval, the high rates of egg production and the extensive infrastructure to fractionate egg yolk proteins, it should be possible to produce large amounts of foreign protein in the eggs of transgenic chickens.

Genetic modification of primordial germ cells by gene trapping, gene targeting, and ϕC31 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 β‐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 ϕC31 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. Mol. Reprod. Dev. 75: 1163–1175, 2008.

Contributions to somatic and germline lineages of chicken blastodermal cells maintained in culture

Chicken blastodermal cells were cultured for 48 hr as explanted intact embryos, as dispersed cells in a monolayer, or with a confluent layer of mouse fibroblasts. The cells were then dispersed and injected into stage X (E‐G&K) recipient embryos that were exposed to 600 rads of irradiation from a 60Co source. Regardless of the conditions in which the cells were cultured, chimeras with contributions to both somatic tissues and the germline were observed. When blastodermal cells were co‐cultured with mouse embryonic fibroblasts, significantly more somatic chimeras were observed and the proportion of feather follicles derived from donor cells was increased relative to that observed following the injection of cells derived from explanted embryos or monolayer cultures. Culture of blastodermal cells in any of the systems, however, yielded fewer chimeras that exhibited reduced contributions to somatic tissues in comparison to the frequency and extent of somatic chimerism observed following injection of freshly prepared cells. Contributions to the germline were observed at an equal frequency regardless of the conditions of culture, but were significantly reduced in comparison to the frequency and rate of germ‐line transmission following injection of cells obtained directly from stage X (E‐G&K) embryos. These data demonstrate that some cells retain the ability to contribute to germline and somatic tissues after 48 hr in culture and that the ability to contribute to the somatic and germline lineages is not retained equally.

Characteristics of Long-Term Cultures of Avian Primordial Germ Cells and Gonocytes

ABSTRACT Avian cell lines derived from germinal crescent primordial germ cells and gonadal gonocytes with long-term proliferative capacity in vitro and their subsequent rates of colonization and germline transmission are described. In general, male cultures proliferate more rapidly than female cultures although both can be developed into cell lines of >2 × 106 cells, at which time, they can be grown indefinitely and a cell bank can be established. All the cell lines injected into embryos transmitted through the germline with the percentage of germline transmission of both male and female cell lines varying from single digits to the high 90s. The derivation of these primordial germ cell and gonadal cell lines and the subsequent robustness of germline transmission validates these cells as suitable for establishment of lines of chickens bearing novel genetic modifications.

Annual variation in the concentrations of circulating hormones in capercaillie (Tetrao urogallus)

Seasonal variation in the levels of immunoreactive lutropin (LH), follitropin (FSH), prolactin (PRL), corticosterone (B), thyroxine (T4), and triiodothyronine (T3) was measured in the plasma of male and female capercaillies (Tetrao urogallus, Galliformes) in captivity (latitude N 67 degrees). In male capercaillies there was an increase in the concentrations of LH and FSH beginning in March and reaching their maxima in May, which correlated with the nesting period. The concentration of plasma PRL increased from the end of April and reached its highest level simultaneously with the rapid fall of plasma LH and FSH concentrations. It remained elevated until August, Plasma T4 level was depressed after levels of plasma FSH and LH had reached their maxima and was correlated to simultaneous elevation of plasma PRL level. No dramatic seasonal changes in plasma T3 level were noted. In the female capercaillie no marked changes in plasma FSH and LH concentrations were observed. Although four of six females laid eggs only one of them managed to terminate its nesting successfully; five eggs hatched. Changes in prolactin concentration in females parallel those in males. No marked variations were observed in plasma corticosterone concentrations. On the basis of these results it seems probable that captive female capercaillie show depressed gonadotrophin secretion, resulting in unsuccessful nesting. On the other hand it has to be emphasized that gonadotrophin assays may not be sensitive enough, especially in the female, to measure LH and FSH in the volumes of plasma put in the assays.