407 AN ATTEMPT TO GENERATE TRANSGENIC CHICKENS BY INJECTION OF EGFP OR YFP GENE-TRANSFECTED BLASTODERMAL CELLS

Abstract

Transgenic chickens have been generated from blastodermal chimeric chickens created by an injection of the foreign DNA-transfected blastodermal cells into recipient embryos. However, there has been no method that allows the efficient foreign DNA transfection into chicken primary cells without using viral vectors. This method using viral vectors has a limitation on the size of DNA that can be transfected, and homologous recombination is not possible with this method. This study was conducted to establish a DNA transfection method using electroporation, which can transfect up to 5 kb DNAs into cells. Embryos at stage X were obtained from freshly laid eggs (200) of Barred Plymouth Rock chickens. Blastodermal cells (105 cells) were then collected from them and transfected with the linearized EGFP or YFP gene by electroporation using a Nucleofector Device (Amaxa, Inc., Gaithersburg, MD, USA). After transfection, the transfected chicken blastodermal cells were injected into White Leghorn recipient embryos to produce chimeric chickens. By 48 h after gene transfection, EGFP or YFP gene expression in the cells was easily observed under a fluorescence microscope. The gene expression rate, however, could not be determined, because the cultured chicken blastodermal cells were not able to separate and it was impossible to load them on the flow cytometer to determine the rate. The blastodermal cells transfected with the EGFP or YFP gene were cultured over night and injected into recipient embryos. The manipulated embryos were cultured ex ovo until they hatched. Dead embryos before hatch were dissected and observed under the fluorescence microscope to determine whether they had fluorescent organs. EGFP or YFP gene expression was detected at several areas: head, somite, inner tissues, and limb bud of embryos, but not germinal tissues. Six female chicks and 10 male chicks were obtained. Five females and 6 males were raised until they were sexually mature. Five female chickens were artificially inseminated with semen of Barred Plymouth Rock males to obtain progeny from donor transfected cells. However, no progeny was obtained. Thus the contribution of donor cells to the germ cells could not be determined, and it was not clear if the expression of EGFP gene or YFP gene was stable. Furthermore, semen could not be collected from six males because of a technical problem, and males were not mated. Although a large number of cells must be used to transfect a foreign gene, the novel gene transfer method in chicken in this study has great significance in the field of transgenic chicken production. Even if the gene expression is transient, this transfer method will enable us not only to integrate more than 5 kb DNA into the avian genome but also to induce homologous recombination in cultured avian cells.