Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

Summary

We demonstrate our approach to finding potential enhancer elements from developmentally regulated genes and evaluating their function through mosaic zebrafish transgenesis.

Abstract

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.

Protocol

1. Selection and cloning of conserved sequences for testing

1. 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.
2. Once we have selected our sequences, we design primers to amplify each sequence from genomic DNA. Primers should be selected to amplify the conserved region plus 30 or more base p....

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Takara LA Taq	Takara Bio Inc	RR02M
pCR8/GW/TOPO TA Cloning Kit	Invitrogen	K2500-20
Gateway LR Clonase II enzyme Mix	Invitrogen	11791-100
Library efficiency competent cells DH5α	Invitrogen	12535-019
Library efficiency competent cells DB3.1	Invitrogen	11782-018
mMESSAGE mMACHINE Kit	Ambion	AM1340
HiSpeed Plasmid Midi Kit	Qiagen	12643
QIAquick PCR Purification Kit	Qiagen	28104
Flaming/Brown Micropipette Puller	Sutter Instrument Co.	P-97
Pneumatic PicoPump	World Precision Instruments, Inc.	SYS-PV820