Construction of transgenic mice carrying enhanced green fluorescent protein gene by seminiferous tubule microinjection.

OBJECTIVE: To study the feasibility of establishing transgenic mice carrying enhanced green flourescent protein (EGFP) gene by means of seminiferous tubule microinjection. METHODS: The vector expressing enhanced green fluorescent protein under the control of human cytomegalovirus immediate-early promoter (pCMV-EGFP) was selected and mixed with liposome in vitro. Microinjection at different doses of the liposome-entrapped plasmid DNA into the seminiferous tubules of male mice at different ages was performed to establish transgenic mice, which were made to mate with female mice at least 40 d after the microinjection. Genomic DNA was extracted from the offspring of the founder mice for PCR and Southern blotting analysis, and the frozen sections of different tissues from 2 of the founders mice were prepared for fluorescence microscopic observation. RESULTS: Among the 41 mice receiving the microinjection, 32 survived and retained their mating ability and fertility, and among their 382 offspring 133 were positive for EGFP DNA as demonstrated by PCR, 15 of which were confirmed by Southern blotting analysis. The age of the mice or the doses of microinjection they received was not shown to impact the integration of EGFP gene, and fluorescence microscopy failed to detect significant EGFP expression in the tissues of the founder mice (P>0.05) in comparison with normal mice. CONCLUSION: Seminiferous tubule microinjection is simple and practicable to implement gene transfer in mice.