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Engineered gene trap transposons for gene identification in vertebrates

Previous work in our laboratory had shown that a gene trap approach to insertional mutagenesis in Xenopus is possible using a method of transgenesis we had developed, called Restriction Enzyme Mediated Insertion (REMI). (Bronchain et al., 1999).

Although we had shown that we could trap genes using this strategy, there were several reasons why it was worth exploring other means of performing insertional mutagenesis in frogs. Firstly, the transgenic technique we were using mostly likely caused damage to host chromosomes, and therefore leading to mutations unrelated to the insertion sites. Secondly, using the REMI method, integrations occurred in large concatemers, which are known to be unstable and making cloning of insertion sites difficult. Thirdly, it was difficult to consider using REMI transgenesis for a large-scale mutagenesis screen. Hence, we decided to combine to combine a gene trap approach to insertional mutagenesis with SB-mediated transposition in vivo, in order to identify mutations in genes in Xenopus tropicalis.

In this strategy we would aim to use mobile DNA elements to insert gene trap vectors into different sites of the genome. Transposons move as single copies, thus eliminating the problem with concatemer insertions. Furthermore one can set up the scheme such that the transpositions occur in vivo, which means that it can easily be scaled-up. The transposon system we decided to employ in this work was Sleeping Beauty (SB). To this end we constructed many gene trap SB transposons. The first two vectors we generated contained the gene trap vectors we had previously used to trap genes in Xenopus (Bronchain et al. (1999). However based on knowledge gained during the course of this work, we continued to modify these vectors with the aim of making them more useful for our insertional mutagenesis strategies.

For example, Partner 1 had shown that transposition efficiency decreases as the size of the transposon increases, and importantly, as the transposon size increases above 4000 base pairs, the efficiency of transposition decreases dramatically. Since our first generation gene trap vectors were over 4000 base pairs in length, we generated a second generation of gene trap vectors with smaller transposon size. These vectors contain around 500 base pairs of Engrailed2 splice acceptor sequences instead of the 2200 base pairs used in the first generation of SB-based gene trap vectors, thus shortening the total size of the transposons by around 1700 base pairs. Another set of experiments performed in the laboratory of Partner 1 showed that the efficiency of transposition increases if both SB elements used contain the left inverted repeats. We therefore generated a third generation of vectors, containing the left inverted repeats on both sides of the SB-based gene trap transposons.

The aim of these alterations were to improve the efficiency of transposition in vivo. The gene trap transposons we have generated could be used in other model organisms, such as the mouse and zebrafish.

Informations connexes

Reported by

University of Cambridge
Wellcome Trust / Cancer Research UK Gurdon Institute
CB2 1QN Cambridge
United Kingdom