지브라물고기 복제 방법에 의한 유전자 동정 및 유전자트랩법 개발
Developing a Gene-trapping Approach for Gene Identification Using Nuclear Transfer in Zebrafish
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초록
이 연구는 gene-trap construct를 가지고 있는 배양세포로부터 trap gene을 확인하고 클로닝한 다음 이러한 세포를 이용하여 복제 지브라물고기를 만들기 위해 수행되어졌다. 본 연구에서 gene-trap과 연관된 복제 지브라물고기가 성공적으로 만들어졌다. 본 실험에서 두 종류의 백터(SA/GFP-TP와 Neo-TP)가 사용되었다. 이들 벡터에 의해 전이된 모든 종류의 세포는 항생제에 의해 선별을 하여 분석에 이용하였다. SA/GFP-TP에 의해 전이된 세포의 경우, 단일세포상에서 GFP 발현도가 낮아 본 연구에서 동물복제에 사용되지 않았으며, Neo-TP에 의해 전이된 세포주가 복제실험에 이용되었다. Neo-TP 세포에 의한 복제실험 결과, 총 1179개의 핵치환 난으로부터 44(3.7%) 개의 배자가 포배기에 도달하였으며, 8(0.8%) 개의 배자가 부화시기에 이르렀다. 그리고 3마리는 성숙단계에 이르렀으며, 이중 1마리에서 정상적으로 gene-trap 전이가 이루어짐을 Southern blot 분석을 통해 확인되었다.
Abstract
This involves identifying and cloning trapped genes from cultured cells carrying the gene-trap constructs and generating cloned zebrafish using these cells for functional study. Gene-trapping studies in gene-trapped cells were carried out in initial and cloned zebrafish carrying gene-trap events were successfully produced based on the nuclear transplantation technique. Two kind of retroviral gene-trap constructs were adopted. The first one(SA/GFP-TP), constructed in my laboratory, carries a GFP reporter gene containing a splicing acceptor and an internal neo gene. The second one(Neo-TP), obtained from Dr. Hicks (Hicks et al., 1997), contains a promoter-less neo gene located in the LTR sequence of a retroviral vector. The infected cells were subjected to drug selection(neomycin treatment) because the two constructs carry the neomycin resistant gene. All those cells survived the neomycin treatment should carry the proviral insertions. For Neo-TP, Isolated DNA from the neomycin-resistant fibroblast cells infected by Neo-TP, was digested with EcoR1 restriction enzyme and transformed into bacteria after ligation. This procedure led to the isolation of seven clones carrying flanking cellular DNA with a typical retroviral integration signature sequence. These clones contained genomic DNA ranging from 1kb to 7kb and sequences of 300-600 bp were obtained from each of the rescued plasmids. Database searching showed that all of them share high homology to zebrafish sequences. For fish cloning using tagged cells, initially, nucleus donors directly selected from a mixture of cells(Neo-TP cells) were used. A total of 44 embryos(3.7%) out of 1179 transplants were reached blastula stage; 8 of these embryos(0.8%) hatched and 3(0.3%) of them survived to adulthood. One out of three lived cloned zebrafish has an amplified fragment and was labeled with 32P.
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