Development of Tol2 transposon mediated gene trap method in zebrafish using MAZ transcription termination site

â—‹Gembu Abe1, Kazuhide Asakawa1,2, Aki Ito1, Ryuichi Fukuda1, Akira Muto1, Pradeep Lal1,2, Hironori Wada3, Koichi Kawakami1

1)National Institute of Genetics, Division of Molecular and Developmental Biology,
2)Department of Genetics, Graduate University for Advanced Studies,
3)PRESTO, Japan Science and Technology Agency

We have been developing a gene trap method in zebrafish using the Tol2 transposon system. The insertions of transposon-based gene trap vector containing the promoter-less reporter gene and a poly(A) signal following a splice acceptor can cause the reporter expression in temporally and spatially restricted patterns. In the previous gene trap screen, we identified more than 500 fish with unique GFP patterns. However, the insertions infrequently caused abnormality rather than our expectation. In these cases the insertion likely permitted the read-through transcription of the trapped gene. To improve the problem of the read-through transcription, we constructed a new gene trap vector, T2GgSAIzGFFM. This gene trap vector has the MAZ transcription termination site following the poly(A) signal. The MAZ site is known to pause Pol II and activate polyadenylation of upstream poly(A) signal. Using this vector, we have identified 143 unique patterns from more than 500 injected fish. We have analyzed 64 lines out of this 143 trap lines and have identified 19 integration sites in the zebrafish genome till date. Among these insertions, 18 insertions were integrated within the Ensemble transcripts (94.7%). This frequency is higher than that of previous construct. These results suggest that the MAZ site could facilitate the expression of the reporter gene and selection of the fish carrying the insertion trapping the transcript. We will analyze the transcripts of the trapped genes in these trap lines and would like to discuss the MAZ function in the gene trapping.