Y 염색체 특이성 DNA분리와 단일 H-Y 항체 개발에 의한 토끼의 수정란 성 감별에 관한 연구 II. PCR을 이용한 Y 염색체 특이성 DNA의 증폭에 의한 토끼 수정란의 성 감별

Studies on Isolaton of Y-specific DNA Marker and Development of Monoclonal H-Y Antibody for Embryo Sexing in Rabbit II. Sex Determination of Rabbit Embryo by PCR Amplified Y-specific DNA

The purpose of this study was to develop the diagnosis techniques for sex determination of rabbit embryos at preimplantation stage. To detect male specific sequences using polymerase chain reaction, two genes functional on sex determination including SRY and ZFX/Y genes were targeted using multiple oligonucleotide primer sets. Three of them for conserved SRY gene were used for appropriate amplification pattern, and then only one primer set #3 proved to be most efficient, showing male-specific strong signal ofamplified sequences. Using this male specific bandsfrom human, cattle, pig and mouse, the gender of rabbit was determined. As an another system for sex determination system, amplified 910bp fragment from ZFX/Y was digested with several restriction endonuclease and showed gender specific restriction fragments only by Hinf I. Using two different system for sex identification of rabbit in this study, blind tests for 17 samples was conducted and showed identical results from two different methods. And then, amplification limit of PCR reaction for template DNA was estimated using various amounts of DNA for both SRY and ZFX/Y systems, resulted as 20pg and 800pg, respectively. With this results, test for gender identification of rabbit embryos were performed using SRY derived amplification system. From total 22 embryos selected for its developmental state 18 were identified as male embryos, showing significant difference from expected sex ratio 1:1. This biased sex ratio was interpreted as to have been caused by the fact, reported by the fact, reported by several researchers, that male embryos develop more rapidly and are more resistant against the in vitro manipulation than female embryos.