CAJ | 학술논문

提高目的基因的转植效率与可遗传效率是转基因鱼研制的关键点之一。本研究利用近年开发的金鱼转座子系统进行转基因斑马鱼的研制,探讨其在转基因鱼上应用的可行性。通过 PCR 方法,改造 Tgf2转座子供体质粒pTgf2-EF1α-eGFP,将肌球蛋白轻链2启动子(mylz2)与红色荧光蛋白基因(RFP)定向插入其中,构建可在肌肉组织特异性表达红色荧光蛋白的Tgf2转座子供体质粒pTgf2-Mylz2-RFP。通过显微注射,将Tgf2转座子供体质粒与Tgf2转座酶mRNA共注射于斑马鱼(Danio ririo)受精卵中,共注射受精卵972粒,出膜后存活的仔鱼803尾,其中携带外源基因的仔鱼为615尾,阳性率为76.6%。转红色荧光蛋白基因斑马鱼首代F0培育至性成熟,将其中的10尾F0斑马鱼分别与野生型斑马鱼进行配对繁殖,其中1个组合产生了体表呈现均匀红色荧光的F1个体,因此RFP在F0的整合率为10%。将红色荧光F1个体培育至性成熟并与野生型鱼配对繁殖, F2的阳性个体占69%。本研究结果说明,由 Tgf2转座子介导的转基因技术,可有效提高目的基因的转植效率与整合效率,在转基因鱼构建以及相关研究中具有开发应用前景。
제고목적기인적전식효솔여가유전효솔시전기인어연제적관건점지일。본연구이용근년개발적금어전좌자계통진행전기인반마어적연제,탐토기재전기인어상응용적가행성。통과 PCR 방법,개조 Tgf2전좌자공체질립pTgf2-EF1α-eGFP,장기구단백경련2계동자(mylz2)여홍색형광단백기인(RFP)정향삽입기중,구건가재기육조직특이성표체홍색형광단백적Tgf2전좌자공체질립pTgf2-Mylz2-RFP。통과현미주사,장Tgf2전좌자공체질립여Tgf2전좌매mRNA공주사우반마어(Danio ririo)수정란중,공주사수정란972립,출막후존활적자어803미,기중휴대외원기인적자어위615미,양성솔위76.6%。전홍색형광단백기인반마어수대F0배육지성성숙,장기중적10미F0반마어분별여야생형반마어진행배대번식,기중1개조합산생료체표정현균균홍색형광적F1개체,인차RFP재F0적정합솔위10%。장홍색형광F1개체배육지성성숙병여야생형어배대번식, F2적양성개체점69%。본연구결과설명,유 Tgf2전좌자개도적전기인기술,가유효제고목적기인적전식효솔여정합효솔,재전기인어구건이급상관연구중구유개발응용전경。
Improving the efficiency of gene transfer and integration is a key objective in transgenic fish research. To evaluate its use for transgenic fish applications, we used the Tgf2 transposon system to construct a transgenic zebrafish line. The Tgf2 transposon donor plasmid pTgf2-EF1α-eGFP was re-constructed by replacement of the transgenic ele-ments, EF1α-eGFP with the myosin light chain 2 promoter (mylz2) and the red fluorescent protein (RFP) gene, mylz2-RFP. The recombinant Tgf2 transposon donor plasmid, pTgf2-Mylz2-RFP was intended to serve as a transposon donor plasmid that specifically expresses red fluorescent protein in muscle tissue. The donor plasmid and the Tgf2 transposase mRNA were co-injected into zebra fish fertilized eggs. A total of 972 eggs were microinjected and 803 lar-vae survived, with a positive rate of 76.6%. The red fluorescence (RFP) gene transgenic zebrafish (F0) were subse-quently cultivated and 10 sexually mature transgenic individuals were mated with wild-type zebrafish, respectively. F1 individuals that exhibited red fluorescence on their body surface were obtained from one of the mating pairs yielding an integration efficiency of 10%. The positive rate of F2 individuals, which were generated by mating the mature trans-genic F1 with the wild-type fish, was 69%. The mature RFP positive F1 individual was then mated with wild-type fish, yielding a 69%positive rate in the F2 generation. Our results demonstrate that the Tgf2 transposon improves the effi-ciency of gene transfer and integration during transgene treatments. Thus, the Tgf2 transposon can be used to construct transgenic fish and in other transgenic fish research.