[Xenopus] Summary of emails from my request for info about I-Sce1 transgenesis

[Jill Johnston]

Here is a brief summary of the answers I got from my emails about I-Sce1 
transgenesis and vector request.**Thank you again to all who replied 
with helpful advice and tips*.
*

If this opens up a discussion about any of the topics covered please 
reply to the forum (xenopus at lists.mbl.edu) rather than to me directly.  
This way everyone can be involved immediately rather than waiting for me 
to forward replies back to the forum.*
*

Thanks*
*

Jill



****

*
Is it best to use 1 or 2 I-Sce1 sites?Should be inverted? Is backbone 
integration a problem?*

The majority of responders use 2 sites but only one said the sites are 
inverted in thevectors his lab uses.Interestingly though, in a very 
recent paper by Shoko Ishibashi, a single sites seems to give the best 
rate of broad expression in F0 embryos.The pTransgenesis vectors that 
have been generated have just a single I-Sce1 site.No one seemed too 
concerned about backbone integration.

*Is it advisable to include insulators in the vector?*

Many responders said yes.The first one mentioned is a tandem HS4 chicken 
b-globin insulator that is placed on either end of the promoter-GOI-SV40 
polyA cassette.This is a small 550 bp sequence whereas the one that is 
included in the pTransgenesis kit (SAR-CH4) is 2.2kb.There is only 1 
insulator in the kit as far as I can tell.I haven't been able to find 
out which of the HS4 or SAR-CH4 is better or why there is only one 
insulator in the pDEST vector of the pTransgenesis kit.

There were a few comments made about the protocol that I found helpful.

Aliquot the I-Sce1 and store them at -80.Aliquots can be stored at -20 
for up to 2 weeks before they become useless in generating transgenic 
animals.

F0 embryos are almost always mosaic unlike those generated using the 
REMI technique.

It is believed that the enzyme stabilizes the cut ends of the injected 
DNA but it is no known how the DNA becomes integrated into the genome.

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