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<p class="MsoNormal">Here is a brief summary of the answers I got
from my emails about I-Sce1 transgenesis and vector request.<b
style=""> </b>Thank you again to all who replied with helpful
advice and tips<b style="">.<br>
</b></p>
<p class="MsoNormal">If this opens up a discussion about any of the
topics covered please reply to the forum (<a class="moz-txt-link-abbreviated" href="mailto:xenopus@lists.mbl.edu">xenopus@lists.mbl.edu</a>)
rather than to me directly. This way everyone can be involved
immediately rather than waiting for me to forward replies back to
the forum.<b style=""><br>
</b></p>
<p class="MsoNormal">Thanks<b style=""><br>
</b></p>
<p class="MsoNormal">Jill</p>
<p class="MsoNormal"><br>
</p>
<p class="MsoNormal"><br>
<b style=""></b><b style=""></b></p>
<p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><br>
Is it best to use 1
or 2 I-Sce1 sites?<span style="mso-spacerun: yes"> </span>Should
be
inverted? Is backbone integration a problem?<o:p></o:p></b></p>
<p class="MsoNormal">The majority of responders use 2 sites but only
one said the
sites are inverted in the<span style="mso-spacerun: yes"> </span>vectors
his
lab uses.<span style="mso-spacerun: yes"> </span>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.<span
style="mso-spacerun:
yes"> </span>The pTransgenesis vectors that have been
generated have just
a single I-Sce1 site.<span style="mso-spacerun: yes"> </span>No
one
seemed too concerned about backbone integration.</p>
<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal"><b style="mso-bidi-font-weight:normal">Is it
advisable to
include insulators in the vector?<o:p></o:p></b></p>
<p class="MsoNormal">Many responders said yes.<span
style="mso-spacerun:
yes"> </span>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.<span style="mso-spacerun: yes"> </span>This is a
small 550 bp sequence whereas
the one that is included in the pTransgenesis kit (SAR-CH4) is
2.2kb.<span style="mso-spacerun: yes"> </span>There is only 1
insulator in the kit as
far as I can tell.<span style="mso-spacerun: yes"> </span>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.</p>
<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal">There were a few comments made about the
protocol that I
found helpful.</p>
<p class="MsoNormal">Aliquot the I-Sce1 and store them at -80.<span
style="mso-spacerun: yes"> </span>Aliquots can be stored at
-20 for up to
2 weeks before they become useless in generating transgenic
animals.</p>
<p class="MsoNormal">F0 embryos are almost always mosaic unlike
those generated
using the REMI technique.</p>
<p class="MsoNormal">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.</p>
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