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한국재래닭(오계) 원시생식세포에 있어 동결방법의 개선이 융해 후생존율에 미치는 영향

The Effect of Modified Cryopreservation Method on Viability of Frozen-thawed Primordial Germ Cell on the Korean Native Chicken(Ogye)

김동훈|김성우|도윤정|한재용|최성복|고응규|성환후|김현
한국축산학회 (Korean Society of Animal Sciences and Technology)|2013.10|한국축산학회지 (Journal of Animal Science and Technology)|제55권 제5호|pp. 427-434

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참고문헌

키워드

생존율ViabilityEG오계원시생식세포Slow freezingPrimordial germ cells(PGCs)Korean native chicken(Ogye)동결방법
주제분류축산학

초록

귀중한 한국재래닭 (오계)의 원시생식세포를 냉동보존하고, 키메라 닭을 통한 재래종의 복원을 도모하는 방법을 실용화하기 위해서, 오계의 원시생식세포에 있어 최적의 동결방법에 대해 검토했다. 원시생식세포의 동결은, 세포를 분리한 후, 동결배지의 기초가 되는 혈청으로서 소 태아혈청 (FBS) 그리고 닭 혈청(CS)를 이용하고, 동결보호제로서는 EG 및 DMSO의 첨가량을 2.5, 5, 10, 15%로 설정하고, 300 μL의 동결배지 용량으로 동결 튜브 안에서 －70℃로 동결했다. 융해 후에는 오계PGC의 생존율을 확인 및 검토를했다. 그 결과, FBS 처리군이 CS 처리군 보다 생존 PGC 회수율이 높은 경향을 나타냈다. 또한 항동해제로서 최적의 조건은 10%EG + FBS의 조합을 기초로 한 동결배지에서 가장 높은 오계원시생식세포의 생존율을 확인했다. 이러한 결과들로부터 한국재래종(오계)의 원시생식세포의 동결보존의 실용화가 보다 더 향상 될 수있는 또 하나의 방법이 될 수 있음을 시사한다.

Abstract

This study was conducted to establish methods for preserving chicken primordial germ cells(PGCs) for long-term storage in liquid nitrogen and for developmental engineering or preservation of species. The purpose of this study is to clarify the effects of fetal bovine serum(FBS) or chicken serum(CS) treatment on the viability of cryopreserved PGCs from Korean Native Chicken (Ogye). PGCs separated from a germinal gonad of an early embryo at day 5.5-6(stage 28) were suspended in a freezing medium containing freezing and protective agents(dimethyl sulfoxide(DMSO), ethylene glycol(EG) and glycerol). The values from 0, 5, 10, and 15 % DMSO plus FBS treatment were 21.6, 30.36, 36.42, 50.39, and 48.36 %, respectively. The viability of PGCs after freeze-thawing was significantly higher for 10% EG plus FBS treatment than for 10% EG+FCS treatment(p<0.05) (64.36% vs. 50.66%). This study establishes a method for preserving chicken PGC that enables systematic storage and labeling of cryopreserved PGC in liquid nitrogen at a germplasm repository and an ease of entry into a database. In the future, the importance for this new technology is that poultry lines can be conserved while work is being conducted to improve the production of germline chimeras.

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