<html><body style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space; ">Dear Jonathan,<div><br></div><div>We contemplated the strategy you suggest, i.e. making a bicistronic transgene with cmlc2:gfp as a transgenesis reporter. We abandoned this strategy because we felt there was a high danger of crosstalk between the two promoter drivers. In your case, the danger would be cmlc2 driving your oncogene and mitfa driving eGfp. So if you don't care if your oncogene might get expressed in the heart, then I guess you wouldn't have to worry. </div><div><br></div><div>I wasn't sure from your posting why you don't think you will be able to tell if the F1 are transgenic without cancer. I mean the mitfa promoter is active from late somitogenesis forward, so it seems to me you should be able to see fluorescence if you have a transgenic. Perhaps you are trying to do this by "transient transgenesis", which is to say looking at effects in the injected generation. If so, my main concern would be neoplastic effects downstream of ectopic expression. We see a lot of ectopic (especially somitic) expression of transgenes in the injected generation when using the Tol2 system, even when we get nice, clean expression of the transgene in the F1 stables. </div><div><br></div><div>Best,</div><div>Wilson</div><div><div apple-content-edited="true"> <span class="Apple-style-span" style="border-collapse: separate; border-spacing: 0px 0px; color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; text-align: auto; -khtml-text-decorations-in-effect: none; text-indent: 0px; -apple-text-size-adjust: auto; text-transform: none; orphans: 2; white-space: normal; widows: 2; word-spacing: 0px; "><div style="word-wrap: break-word; -khtml-nbsp-mode: space; -khtml-line-break: after-white-space; "><span class="Apple-style-span" style="border-collapse: separate; border-spacing: 0px 0px; color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; text-align: auto; -khtml-text-decorations-in-effect: none; text-indent: 0px; -apple-text-size-adjust: auto; text-transform: none; orphans: 2; white-space: normal; widows: 2; word-spacing: 0px; "><span class="Apple-style-span" style="border-collapse: separate; border-spacing: 0px 0px; color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; text-align: auto; -khtml-text-decorations-in-effect: none; text-indent: 0px; -apple-text-size-adjust: auto; text-transform: none; orphans: 2; white-space: normal; widows: 2; word-spacing: 0px; "><span class="Apple-style-span" style="border-collapse: separate; border-spacing: 0px 0px; color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; text-align: auto; -khtml-text-decorations-in-effect: none; text-indent: 0px; -apple-text-size-adjust: auto; text-transform: none; orphans: 2; white-space: normal; widows: 2; word-spacing: 0px; "><span class="Apple-style-span" style="border-collapse: separate; border-spacing: 0px 0px; color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; text-align: auto; -khtml-text-decorations-in-effect: none; text-indent: 0px; -apple-text-size-adjust: auto; text-transform: none; orphans: 2; white-space: normal; widows: 2; word-spacing: 0px; "><span class="Apple-style-span" style="border-collapse: separate; border-spacing: 0px 0px; color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; text-align: auto; -khtml-text-decorations-in-effect: none; text-indent: 0px; -apple-text-size-adjust: auto; text-transform: none; orphans: 2; white-space: normal; widows: 2; word-spacing: 0px; "><span class="Apple-style-span" style="border-collapse: separate; border-spacing: 0px 0px; color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; text-align: auto; -khtml-text-decorations-in-effect: none; text-indent: 0px; -apple-text-size-adjust: auto; text-transform: none; orphans: 2; white-space: normal; widows: 2; word-spacing: 0px; "><span class="Apple-style-span" style="border-collapse: separate; border-spacing: 0px 0px; color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; text-align: auto; -khtml-text-decorations-in-effect: none; text-indent: 0px; -apple-text-size-adjust: auto; text-transform: none; orphans: 2; white-space: normal; widows: 2; word-spacing: 0px; "><div>--------------------------------------------------------------------------------------------</div><div>Wilson Clements, Ph.D. </div><div><br class="khtml-block-placeholder"></div><div><a href="mailto:wclements@ucsd.edu">wclements@ucsd.edu</a></div><div><br class="khtml-block-placeholder"></div><div>Dept. of Biology </div><div>Section of Cell and Developmental Biology </div><div>University of California at San Diego </div><div>9500 Gilman Dr.</div><div>Natural Sciences Building 6105</div><div>La Jolla, CA 92093-0380</div><div><br class="khtml-block-placeholder"></div><div>TEL (858) 534-6955</div>LAB (858) 822-4658<br class="Apple-interchange-newline"></span></span><div>FAX (858) 822-5740</div><br class="Apple-interchange-newline"></span></span></span></span></span></div></span> </div><div><br><div>Begin forwarded message:</div><br class="Apple-interchange-newline"><blockquote type="cite"><div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; "><font face="Helvetica" size="3" color="#000000" style="font: 12.0px Helvetica; color: #000000"><b>From: </b></font><font face="Helvetica" size="3" style="font: 12.0px Helvetica"><a href="mailto:zbrafish-request@oat.bio.indiana.edu">zbrafish-request@oat.bio.indiana.edu</a></font></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; "><font face="Helvetica" size="3" color="#000000" style="font: 12.0px Helvetica; color: #000000"><b>Date: </b></font><font face="Helvetica" size="3" style="font: 12.0px Helvetica">February 27, 2009 9:03:32 AM PST</font></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; "><font face="Helvetica" size="3" color="#000000" style="font: 12.0px Helvetica; color: #000000"><b>To: </b></font><font face="Helvetica" size="3" style="font: 12.0px Helvetica"><a href="mailto:zbrafish@magpie.bio.indiana.edu">zbrafish@magpie.bio.indiana.edu</a></font></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; "><font face="Helvetica" size="3" color="#000000" style="font: 12.0px Helvetica; color: #000000"><b>Subject: </b></font><font face="Helvetica" size="3" style="font: 12.0px Helvetica"><b>Zbrafish Digest, Vol 45, Issue 16</b></font></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; "><font face="Helvetica" size="3" color="#000000" style="font: 12.0px Helvetica; color: #000000"><b>Reply-To: </b></font><font face="Helvetica" size="3" style="font: 12.0px Helvetica"><a href="mailto:zbrafish@oat.bio.indiana.edu">zbrafish@oat.bio.indiana.edu</a></font></div><div style="margin-top: 0px; margin-right: 0px; margin-bottom: 0px; margin-left: 0px; min-height: 14px; "><br></div> </div><div>Send Zbrafish mailing list submissions to<br><span class="Apple-tab-span" style="white-space:pre"> </span><a href="mailto:zbrafish@net.bio.net">zbrafish@net.bio.net</a><br><br>To subscribe or unsubscribe via the World Wide Web, visit<br><span class="Apple-tab-span" style="white-space:pre"> </span>http://www.bio.net/biomail/listinfo/zbrafish<br>or, via email, send a message with subject or body 'help' to<br><span class="Apple-tab-span" style="white-space:pre"> </span>zbrafish-request@net.bio.net<br><br>You can reach the person managing the list at<br><span class="Apple-tab-span" style="white-space:pre"> </span>zbrafish-owner@net.bio.net<br><br>When replying, please edit your Subject line so it is more specific<br>than "Re: Contents of Zbrafish digest..."<br><br><br>Today's Topics:<br><br> 1. Zfish injection/integration control (Jonathan Schumacher)<br><br><br>----------------------------------------------------------------------<br><br>Message: 1<br>Date: Thu, 26 Feb 2009 11:10:10 -0800 (PST)<br>From: Jonathan Schumacher <jaschumac@gmail.com><br>Subject: [Zbrafish] Zfish injection/integration control<br>To: bionet-organisms-zebrafish@moderators.isc.org<br>Message-ID:<br><span class="Apple-tab-span" style="white-space:pre"> </span><ea171c1d-a725-4f94-9b99-c507f3a7fbd3@r22g2000vbp.googlegroups.com><br>Content-Type: text/plain; charset=ISO-8859-1<br><br>Greetings,<br><br>I have designed a construct of MITFA driving my oncogene and<br>cytoplasmic-expressed EGFP, subsequently cloned into the pDest<br>Tol2pA. We suspect that it will take at least 8 weeks to see whether<br>the fish develop cancer and after setting up injections, I realized<br>that I have no injection/integration control. My question is would<br>you suggest recloning my construct into the pDest Tol2pA with cmlc2-<br>driven EGFP? I believe that this would lead to double-EGFP labeling<br>and am unclear if this would be problematic. Is there an alternative<br>strategy, like coinjecting my destination vector with a cherry-labeled<br>cmlc2 (or other)?<br><br>Your thoughts & suggestions are greatly appreciated.<br><br>Best,<br><br>Jonathan<br><br><br><br>------------------------------<br><br>_______________________________________________<br>Zbrafish mailing list<br>Zbrafish@net.bio.net<br>http://www.bio.net/biomail/listinfo/zbrafish<br><br>End of Zbrafish Digest, Vol 45, Issue 16<br>****************************************<br><br></div></blockquote></div><br></div></body></html>
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