[Xenopus] Summary of emails from my request for info about I-Sce1 transgenesis
johnstoj at ucalgary.ca
Sun Apr 15 13:48:23 EDT 2012
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.*
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
F0 embryos are almost always mosaic unlike those generated using the
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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