Detection of the EGFP sequence in breast muscle of 3-year-old chicken after transfection using sonoporation.

In our continuing effort to generate transgenic chickens, sonoporation was chosen to insert an exogenous gene into the chicken genome. An EGFP expression vector (pCAG-EGFPac) and microbubbles were injected into the central disc of stage-X blastoderm or the germinal crescent of stage-4 embryos, followed by ultrasonic vibration. Nineteen chicks out of 108 treated embryos hatched, six females and six males out of these 19 chicks grew to sexual maturity and two females and three males lived for 3 years. Genomic DNA from 17 out of 35 gonads from embryos and chicks that died before sexual maturity was EGFP-positive by PCR. No EGFP sequence was detected in the genomic DNA of 322 embryos from six sexually mature females and the semen from four sexually mature males by PCR. When genomic DNA was obtained from various tissues of five 3-year-old chickens, the EGFP sequence was amplified from the genomic DNA of the breast muscle of a female (No. 85). The above sequence was subjected to DNA sequencing and verified to be the EGFP sequence. These results showed that sonoporation is an effective tool for the transduction of exogenous genes into chicken embryos for the generation of transgenic chickens.