[Xenopus] I-Sce1 transgenesis plasmids

[Jill Johnston]

We want to try using the I-Sce1 protocol for making transgenic laevis 
but have some questions about plasmids design.

Some labs use vectors with one I-Sce1 site and others use vectors with 2 
inverted I-Sce1 sites.  Is one better than the other?  I am confused 
about how the inverted site vectors would work.  Wouldn't the genomic 
DNA have to be cut in 2 places for this to work since the sticky ends of 
the insert are not complementary? Also if you have the plasmid cut in 2 
pieces don't you run the risk of having the vector backbone becoming 
integrated into the genome?

Is it advisable to use insulators on either side of the 
promoter-gene-SV40 cassette?

Thank you

Jill Johnston
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