Generation of Sperms Containing EGFP-LacZ Following Transfection of Chicken Testis with a Eukaryotic Dual Reporter Vector

Abstract

Male germ cells modified by foreign genes can be used to generate transgenic chicken. In this study, in vivo transfection of chicken testis with an EGFP-LacZ dual reporter expression vector was performed. Large-scale plasmid DNA preparation of the EGFP-LacZ eukaryotic expression vector was carried out and efficient transfection of chicken testicle cells using the prepared plasmid DNA was confirmed in vitro. The reporter plasmid was directly injected into adult rooster testes. Semen samples were collected on 10-days post-transfection and every other day thereafter; and a total of six collections were made. Semen slides were subjected to fluorescence microscopy and β-galactosidase activity assay to identify sperms carrying the reporter genes. The presence of EGFP and LacZ was further confirmed by PCR amplification with sperm genomic DNA as template. The testicles of those birds were subjected to cryostat sectioning, fluorescence microscopy and β-galactosidase activity assay. The results showed that sperms with green fluorescence were not observed on semen slides; however, sperms positive for β-galactosidase were detected. Specific amplicons of EGFP and lacZ were detected in four of the six sequentially collected semen samples. Fluorescence microscopy of the corresponding semen slides revealed yellow-green fluorescence, but not clear green fluorescence. The β-galactosidase activity assay and GFP histochemistry using monoclonal antibodies demonstrated positive staining for subsets of testicle cells. Together, these results showed that direct injection of the dual reporter vector into adult rooster testis allowed in vivo transfection of chicken sperm precursor cells, which further developed into sperm containing EGFP-LacZ.