Name: mosaic zebrafish transgenesis for evaluating enhancer sequences
Uploaded: 2010-07-16T17:00:00
Description: Watch this Scientific Journal Video about Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences at JoVE.com

<p class="jove_content">We thank Ms. Paula Roy for excellent fish husbandry, and Steve Ekker for the kind gift of the pDB600 plasmid. These studies were funded by a grant to S.F. from NHGRI.</p>

<p class="jove_title">1. Selection and cloning of conserved sequences for testing</p>
<ol>
  <li> One must first identify potential regulatory sequences for functional testing. We rely on evolutionary sequence conservation as a criterion to select sequences, and most often use the algorithm PhastCons, which is accessible as a track on the UCSC Genome browser (http://genome.ucsc.edu/cgi-bin/hgGateway). However, there are many other algorithms available to evaluate sequence conservation across species.</li>
  <li> Once we have selected our seq...

<ol>
	<li>Fisher, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+biological+relevance+of+putative+enhancers+using+Tol2+transposon-mediated+transgenesis+in+zebrafish.">Evaluating the biological relevance of putative enhancers using Tol2 transposon-mediated transgenesis in zebrafish.</a> <em style='font-style: italic'>Nat Protoc</em>. <b>1</b> (3), 1297-12 (2006).</li><li>Balciunas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+a+high+cargo-capacity+transposon+for+genetic+applications+in+vertebrates.">Harnessing a high cargo-capacity transposon for genetic applications in vertebrates.</a> <em style='font-style: italic'>PLoS Genet</em>. <b>2</b> (11), e169-169 (2006).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> <em style='font-style: italic'>The Zebrafish Book</em>. , (1995).</li></ol>

<p class="jove_content">We have demonstrated the approach used in our laboratory for the efficient analysis of potential enhancers using zebrafish transgenesis. Most often, we use this approach to look for tissue-specific enhancers associated with human genes. Despite the lack of obvious sequence homology most of the time between human and fish non-coding sequences, we find that this approach is usually successful in identifying enhancers for the genes of interest to us. The critical factors for success are taking care in the preparation of the...

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<td>Takara LA Taq</td>
<td>&nbsp;</td>
<td>Takara Bio Inc</td>
<td>RR02M</td>
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<td>pCR8/GW/TOPO TA Cloning Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>K2500-20</td>
<td>&nbsp;</td>
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<td>Gateway LR Clonase II enzyme Mix</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11791-100</td>
<td>&nbsp;</td>
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<td>Library efficiency competent cells DH5&alpha;</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12535-019</td>
<td>&nbsp;</td>
<tr>
<td>Library efficiency competent cells DB3.1</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11782-018</td>
<td>&nbsp;</td>
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<td>mMESSAGE mMACHINE Kit</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AM1340</td>
<td>&nbsp;</td>
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<td>HiSpeed Plasmid Midi Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>12643</td>
<td>&nbsp;</td>
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<td>QIAquick PCR Purification Kit </td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>28104</td>
<td>&nbsp;</td>
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<td>Flaming/Brown Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instrument</td>
<td>P-97</td>
<td>&nbsp;</td>
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<td>Pneumatic PicoPump</td>
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<td>World Precision Instruments</td>
<td>SYS-PV820</td>
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mosaic zebrafish transgenesis for evaluating enhancer sequences

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non coding sequences. One prominent function carried out by the non coding fraction of the genome is to regulate gene transcription; however, there are no effective methods to broadly predict cis-regulatory elements from primary DNA sequence. We have developed an efficient protocol to functionally evaluate potential cis-regulatory elements through zebrafish transgenesis. Our approach offers significant advantages over cell-culture based techniques for developmentally important genes, since it provides information on spatial and temporal gene regulation. Conversely, it is faster and less expensive than similar experiments in transgenic mice, and we routinely apply it to sequences isolated from the human genome. Here we demonstrate our approach to selecting elements for testing based on sequence conservation and our protocol for cloning sequences and microinjecting them into zebrafish embryos.

Watch this Scientific Journal Video about Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences at JoVE.com

Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

<p class="jove_content">We demonstrate our approach to finding potential enhancer elements from developmentally regulated genes and evaluating their function through mosaic zebrafish transgenesis.</p>

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non ...

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