ABSTRACT
<p><b>AIM</b>To confirm the stability of exogenous genes in the generation of transgenic chickens using ejaculated chicken sperm, the deoxyribonuclease (DNase) activity was evaluated in the seminal plasma of ejaculated semen and the stability of DNA was examined by adding lipofection reagents.</p><p><b>METHODS</b>A PCR fragment (249 bp) of pEGFPN-1 vector was used as the DNA substrate and was incubated with the seminal plasma at 40 degree C for 30 min. Then, the whole reaction solution was subjected to agarose gel electrophoresis and the DNA size was evaluated under UV light.</p><p><b>RESULTS</b>The DNA substrate was completely diminished after incubation with seminal plasma. However, the substrate was intact after incubation with heat-treated seminal plasma or incubation with seminal plasma in the presence of 0.5 mmol/L approximately 5 mmol/L EDTA. The substrate was stabilized in the seminal plasma by the addition of commercially available lipofection reagents.</p><p><b>CONCLUSION</b>The DNase activity is present in the seminal plasma of ejaculated chicken semen. However, DNA is stable in the liposomal-DNA complex.</p>
Subject(s)
Animals , Male , Cations, Divalent , Pharmacology , Chickens , Physiology , DNA , DNA Primers , Deoxyribonucleases , Edetic Acid , Pharmacology , Hot Temperature , Indicators and Reagents , Reverse Transcriptase Polymerase Chain Reaction , SemenABSTRACT
<p><b>AIM</b>The production of interspecific germline chimeras between chicken and quail were attempted employing the dissociated cells derived from the blastodermal central disk (stage X) and the germinal crescent region of embryo (stage 7-8).</p><p><b>METHODS</b>The central disk (CD) of the area pellucida in chicken blastoderm (stage X) and the germinal crescent region (GCR) of embryo (stage 7-8) were dispersed and injected into the subgerminal cavity of quail blastoderm (stage X). Injected eggs were incubated for 7 days or to hatching. The donor chicken DNA was detected by the polymerase chain reaction.</p><p><b>RESULTS</b>In day-7 embryos, chicken DNA was detected in 5 gonads and 9 brains from 53 survived embryos received chicken CD cells, and 1 gonads and 6 brains from 27 survived embryos received chicken GCR. Chicken DNA was also detected from the semen of one adult male hatched from eggs received chicken GCR cells.</p><p><b>CONCLUSION</b>CD and GCR cells as the donors showed the possibility to produce the interspecific germline chimera, but further studies are needed to make necessary improvement.</p>