Molecular Biotechnology: Principles and Applications - Glick, B., Pasternak, J. 2002

Molecular Biotechnology of Microbiological Systems
Transgenic Animals
Transgenic Birds

Microinjection of DNA into fertilized avian eggs to establish transgenic lines is a challenging Procedure, largely due to specific aspects of bird reproduction and development. Unlike mammals, where typically a single spermatozoon enters the egg upon Fertilization, avian polyspermy frequently occurs, making it impossible to identify which male pronucleus will fuse with the female one. Cytoplasmic DNA microinjection is likewise unsuitable, as the DNA fails to integrate into The Genome of the fertilized ovum. Finally, even if nuclear microinjection is successful, subsequent manipulations remain difficult because the avian zygote is rapidly encased in a tough membrane, followed by an albumin layer and both inner and outer calcareous shells.

However, the transgene can be introduced into the yolk region (the germinal disc), which contains both male and female pronucleuses and forms prior to shell deposition. Following DNA delivery, each egg is cultured in vitro until an embryo develops, at which point it is transferred into a surrogate egg to simulate hatching. A single line of transgenic chicks has been successfully produced using this strategy; nonetheless, the method remains inefficient and technically demanding under standard laboratory conditions. By the time the outer calcareous shell of the avian egg hardens, the embryo at the blastoderm stage already consists of two layers comprising 40,000 and 80,000 Cells, respectively. Experiments have been conducted involving the inoculation of such embryos with Replication-defective retroviral vectors carrying bacterial marker genes. This approach yielded transgenic chickens and common quail harboring foreign genes in their germline cells. Although such birds generally do not produce free Viral Particles, The Use of retroviral vectors as delivery vehicles for foreign genes in animals intended for human consumption inevitably raises safety concerns. Furthermore, the size of the transgene that can be introduced via a retroviral vector does not exceed ~8 kb, and integration into the initial insertion site is occasionally unstable. These limitations have driven researchers to pursue alternative Methods of transgenesis.

Class="center">

Fig. 19.14. Production of transgenic chickens via transfection of isolated blastoderm cells. Isolated cells are transfected with a transgene using Liposomes and introduced into the subgerminal cavity of an irradiated recipient blastoderm. Some of the resulting offspring are chimeras, and those carrying the transgene in their germline cells can give rise to transgenic lines upon breeding.

Since no bird-specific ES cells have been discovered, ES Cell-based approaches are not applicable to avian species. A more promising avenue relies on recombinant embryonic cells. The procedure is as follows: blastoderm cells are isolated from a chicken embryo, transfected using cationic Lipids (liposomes) complexed with transgenic DNA (lipofection), and reinjected into the subgerminal cavity of freshly laid eggs (Fig. 19.14). A fraction of the offspring will retain donor cells in varying small amounts; such animals are termed chimeras. In some chimeras, cells derived from the transfected population may contribute to the germline, and following several rounds of crossbreeding, transgenic animal lines can be established. To increase the probability of generating chimeras carrying foreign genes in their germline, the proportion of donor cells can be enhanced by irradiating recipient embryos prior to cell transfer (540–660 rad for 1 h). This irradiation causes some—though not all—blastoderm cells to perish, shifting The ratio of transfected to recipient cells in favor of the former. While efficiency remains low, this approach appears viable for producing transgenic chicks.

Transgenic chickens can be utilized to improve the genotypes of existing breeds—specifically, to confer in vivo resistance to VIRAL INFECTIONS AND coccidiosis, enhance feed conversion efficiency, reduce fat and Cholesterol levels in eggs, and improve meat quality. Furthermore, it has been proposed to harness the high protein content of avian eggs as a bioreactor system for the pharmaceutical industry. Targeting transgene expression to the reproductive tract of hens, where large quantities of Ovalbumin are normally secreted, could facilitate the accumulation of valuable recombinant Proteins in the egg, from which they can subsequently be purified.