In Vivo Gene Transfer into Chicken Embryos via Primordial Germ Cells Using Green Fluorescent Protein as a Marker.

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  • Ebara Fumio
    Animal Resource Science Section, Division of Bioresource and Bioenvironmental Sciences, Graduate School, Kyushu University, Fukuoka 812-8581, Japan
  • Fujihara Noboru
    Animal Resource Science Section, Division of Bioresource and Bioenvironmental Sciences, Graduate School, Kyushu University, Fukuoka 812-8581, Japan

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An exogenous gene (GFP&lacZ/pkkv4-lacZ) introduced into the germinal crescent region (GCR) of avian embryos was confirmed to be successfully transferred to the gonads via the primordial germ cells (PGCs). The hatched chicks were raised until the stage of sexual maturation. The incorporation of pkkv4-lacZ was detected in both male and female DNA-treated chickens (DNA-chicken). Female DNA-chicken were subjected to artificial insemination using sperm from normal male chickens according to routine methods. Fertilized eggs obtained from female DNA-chicken were incubated for 72 h and the expression of green fluorescent protein (GFP) gene in the embryos of offspring was detected under a fluorescent microscope. Following the examination for GFP the embryos removed from the yolk were also examined by X-gal staining to detect the expression of the lacZ gene in offspring, and the expression of pkkv4-lacZ was clearly detected. Finally, the presence of pkkv4-lacZ in the extracts from embryos was determined by polymerase chain reaction (PCR) analysis. In male DNA-chicken, the presence of injected DNA was also confirmed in the extracts from sperm. These results suggest that the exogenous gene introduced into the GCR of chicken embryos migrated successfully to the gonad, resulting in incorporation into the offspring and spermatozoa of DNA-chicken.

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