Helen Sang

Efficient production of germline transgenic chickens using lentiviral vectors

An effective method for genetic modification of chickens has yet to be developed. An efficient technology, enabling production of transgenic birds at high frequency and with reliable expression of transgenes, will have many applications, both in basic research and in biotechnology. We investigated the efficiency with which lentiviral vectors could transduce the chicken germ line and examined the expression of introduced reporter transgenes. Ten founder cockerels transmitted the vector to between 4% and 45% of their offspring and stable transmission to the G2 generation was demonstrated. Analysis of expression of reporter gene constructs in several transgenic lines showed a conserved expression profile between individuals that was maintained after transmission through the germ line. These data demonstrate that lentiviral vectors can be used to generate transgenic lines with an efficiency in the order of 100‐fold higher than any previously published method, with no detectable silencing of transgene expression between generations.

The Oct4 homologue PouV and Nanog regulate pluripotency in chicken embryonic stem cells

Embryonic stem cells (ESC) have been isolated from pregastrulation mammalian embryos. The maintenance of their pluripotency and ability to self-renew has been shown to be governed by the transcription factors Oct4 (Pou5f1) and Nanog. Oct4 appears to control cell-fate decisions of ESC in vitro and the choice between embryonic and trophectoderm cell fates in vivo. In non-mammalian vertebrates, the existence and functions of these factors are still under debate, although the identification of the zebrafish pou2 (spg; pou5f1) and Xenopus Pou91 (XlPou91) genes, which have important roles in maintaining uncommitted putative stem cell populations during early development, has suggested that these factors have common functions in all vertebrates. Using chicken ESC (cESC), which display similar properties of pluripotency and long-term self-renewal to mammalian ESC, we demonstrated the existence of an avian homologue of Oct4 that we call chicken PouV (cPouV). We established that cPouV and the chicken Nanog gene are required for the maintenance of pluripotency and self-renewal of cESC. These findings show that the mechanisms by which Oct4 and Nanog regulate pluripotency and self-renewal are not exclusive to mammals.

Prospects for transgenesis in the chick

Research to develop a useful method for genetic modification of the chick has been on-going since the first demonstrations in the mouse in the 1980s that genetic modification is an invaluable tool for the study of gene function. Manipulation of the chick zygote is possible but inefficient. Considerable progress has been made in developing potentially pluripotent embryo stem cells and their contribution to somatic chimeric birds well-established. Germ line transmission of gametes derived from genetically modified embryo cells has not been described. Transfer of primordial germ cells from a donor embryo to a recipient and production of functional gametes from the donor-derived cells is possible. Genetic modification of primordial germ cells before transfer and their recovery through the germ line has not been achieved. The first transgenic birds described were generated using retroviral vectors. The use of lentiviral vectors may make this approach a feasible method for transgenic production, although there are limitations to the applications of these vectors. It is likely that a method will be developed in the next few years that will enable the use of transgenesis as a tool in the study of development in the chick and for many other applications in basic research and biotechnology.

Transgenic chickens as bioreactors for protein-based drugs

The potential of using transgenic animals for the synthesis of therapeutic proteins was suggested over twenty years ago. Considerable progress has been made in developing methods for the production of transgenic animals and specifically in the expression of therapeutic proteins in the mammary glands of cows, sheep and goats. Development of transgenic hens for protein production in eggs has lagged behind these systems. The positive features associated with the use of the chicken in terms of cost, speed of development of a production flock and potentially appropriate glycosylation of target proteins have led to significant advances in transgenic chicken models in the past few years.

Rapid induction of pluripotency genes after exposure of human somatic cells to mouse ES cell extracts

The expression of 4 pluripotency genes (Oct4, Sox2, c-Myc and Klf4) in mouse embryonic fibroblasts can reprogramme them to a pluripotent state. We have investigated the expression of these pluripotency genes when human somatic 293T cells are permeabilized and incubated in extracts of mouse embryonic stem (ES) cells. Expression of all 4 genes was induced over 1-8 h. Gene expression was associated with loss of repressive histone H3 modifications and increased recruitment of RNA polymerase II at the promoters. Lamin A/C, which is typically found only in differentiated cells, was also removed from the nuclei. When 293T cells were returned to culture after exposure to ES cell extract, the expression of the pluripotency genes continued to rise over the following 48 h of culture, suggesting that long-term reprogramming of gene expression had been induced. This provides a methodology for studying the de-differentiation of somatic cells that can potentially lead to an efficient way of reprogramming somatic cells to a pluripotent state without genetically altering them.

Transgenic chickens--methods and potential applications.

The development of techniques for the genetic manipulation of poultry has lagged behind the technology available in mammalian systems, although several different approaches are being taken to overcome the problems associated with the manipulation of avian embryos. Several methods being developed for generating transgenic chickens are giving promising results, and the production of transgenic chickens by DNA microinjection has recently been demonstrated. Exploitation of this technology, in both basic and applied research, is now a possibility, and many applications of transgenic technology to poultry breeding and novel uses of transgenic chickens have been suggested.

Regulation of Chicken Gonadotropin-Releasing Hormone-I mRNA in Incubating, Nest-Deprived and Laying Bantam Hens

Secretion of luteinizing hormone is decreased when hens start to incubate their eggs and is increased after nest deprivation or hatching of the eggs. The purpose of this study was to determine whether decreased luteinizing hormone (LH) secretion during incubation in the domestic hen is associated with a decrease in hypothalamic chicken gonadotropin-releasing hormone-I (cGnRH-I) mRNA or peptide. A semiquantitative competitive PCR assay was developed to measure cGnRH-I mRNA. Hypothalamic mRNA was quantified as the amount of GnRH cDNA obtained by reverse transcription of cGnRH-I mRNA. The amount of hypothalamic cGnRH-I mRNA was significantly higher in laying than in incubating hens (38.7 +/- 10.3 vs. 7.7 +/- 1.6 x 10(-17) mol cDNA, p = 0.01, n = 8). The hypothalamic GnRH peptide content was not significantly different between laying and incubating hens in either the preoptic area (286.9 +/- 24.01 vs. 269.3 +/- 29.3 pg, n = 8) or the basal hypothalamus (1.67 +/- 0.19 vs. 1.54 +/- 0.21 ng, n = 8). Five days after incubating hens were deprived of their eggs, the resulting increase in LH secretion was associated with a significant increase in hypothalamic content of cGnRH-I mRNA (22.8 +/- 2.2 vs. 6.7 +/- 1.7 x 10(-17) mol cDNA, p < 0.001, n = 8). These observations suggest that a decrease in the expression of the cGnRH-I gene is a major factor in maintaining depressed LH secretion in incubating domestic chickens.

Evidence that Hoxa expression domains are evolutionarily transposed in spinal ganglia, and are established by forward spreading in paraxial mesoderm

Transposition of anatomical structures along the anteroposterior axis has been a commonly used mechanism for changing body proportions during the course of evolutionary time. Earlier work (Gaunt, S.J., 1994. Conservation in the Hox code during morphological evolution. Int. J. Dev. Biol. 38, 549-552; Burke, A.C., Nelson, C.E., Morgan, B.A., Tabin, C., 1995. Hox genes and the evolution of vertebrate axial morphology. Development 121, 333-346) showed how transposition in mesodermal derivatives (vertebrae) could be attributed to transposition in the expression of Hox genes along the axial series of somites. We now show how transposition in the segmental arrangement of the spinal nerves can also be correlated with shifts in the expression domains of Hox genes. Specifically, we show how the expression domains of Hoxa-7, a-9 and a-10 in spinal ganglia correspond similarly in both mouse and chick with the positions of the brachial and lumbosacral plexuses, and that this is true even though the brachial plexus of chick is shifted posteriorly, relative to mouse, by seven segmental units. In spite of these marked species differences in the boundaries of Hoxa-7 expression, cis regulatory elements located up to 5 kb upstream of the chick Hoxa-7 gene showed much functional and structural conservation with those described in the mouse (Puschel, A.W., Balling, R., Gruss, P., 1991. Separate elements cause lineage restriction and specify boundaries of Hox-1.1 expression. Development 112, 279-287; Knittel, T., Kessel, M., Kim, M.H., Gruss, P., 1995. A conserved enhancer of the human and murine Hoxa-7 gene specifies the anterior boundary of expression during embryonal development. Development 121, 1077-1088). We also show that chick Hoxa-7 and a-10 expression domains spread forward into regions of somites that are initially negative for the expression of these genes. We discuss this as evidence that Hox expression in paraxial mesoderm spreads forward, as earlier found for neurectoderm and lateral plate mesoderm, in a process that occurs independently of cell movement.

Transgenesis sunny-side up.

The use of primordial germ cells to transmit genetic modifications through the chicken germ line is a significant advance for avian biology.