We thank: Nicolas Gompel and Benjamin Prud'homme for their contributions to the development of this protocol; Melissa Williams and four anonymous reviewers for comments on the manuscript; the University of Dayton Graduate School for research fellowships to WAR; and the University of Dayton Biology Department and Research Institute (UDRI) for research support for TMW. This work was supported by the American Heart Association Grant 11BGIA7280000 to TMW.

Overview:
This video demonstrates a protocol capable of quantitatively measuring the gene regulatory activities for cis-regulatory element (CRE) sequences in Drosophila (D.) melanogaster. This protocol can be used to compare the regulatory activities possessed by: wild type and mutant CRE forms, naturally-occurring CRE alleles found within a species, or orthologous CREs between diverged species.
1. Site-specific integration of reporter transgenes into the Dr...

<ol>
	<li>Davidson, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Regulatory+Genome.">The Regulatory Genome.</a> Gene Regulatory Networks in Development and Evolution. , (2006).</li><li>Wray, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolutionary+significance+of+cis-regulatory+mutations.">The evolutionary significance of cis-regulatory mutations.</a> Nat. Rev. Genet. 8, 206-216 (2007).</li><li>Stern, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+developmental+biology+and+the+problem+of+variation.">Evolutionary developmental biology and the problem of variation.</a> Evolution. 54, 1079-1091 (2000).</li><li>Carroll, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evo-devo+and+an+expanding+evolutionary+synthesis:+a+genetic+theory+of+morphological+evolution.">Evo-devo and an expanding evolutionary synthesis: a genetic theory of morphological evolution.</a> Cell. 134, 25-36 (2008).</li><li>Sethupathy, P., Collins, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+target+site+polymorphisms+and+human+disease.">MicroRNA target site polymorphisms and human disease.</a> Trends Genet. 24, 489-497 (2008).</li><li>Visel, A., Rubin, E. M., Pennacchio, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+views+of+distant-acting+enhancers.">Genomic views of distant-acting enhancers.</a> Nature. 461, 199-205 (2009).</li><li>Spradling, A. C., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposition+of+cloned+P+elements+into+Drosophila+germ+line+chromosomes.">Transposition of cloned P elements into Drosophila germ line chromosomes.</a> Science. 218, 341-347 (1982).</li><li>Bischof, J., Maeda, R. K., Hediger, M., Karch, F., Basler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+transgenesis+system+for+Drosophila+using+germ-line-specific+phiC31+integrases.">An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases.</a> Proc. Natl. Acad. Sci. U. S. A. 104, 3312-3317 (2007).</li><li>Groth, A. C., Fish, M., Nusse, R., Calos, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+transgenic+Drosophila+by+using+the+site-specific+integrase+from+phage+phiC31.">Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31.</a> Genetics. 166, 1775-1782 (2004).</li><li>Venken, K. J., He, Y., Hoskins, R. A., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P[acman]:+a+BAC+transgenic+platform+for+targeted+insertion+of+large+DNA+fragments+in+in+D.+melanogaster.">P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in in D. melanogaster.</a> Science. 314, 1747-1751 (2006).</li><li>Markstein, M., Pitsouli, C., Villalta, C., Celniker, S. E., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes. , (2008).</li><li>Rebeiz, M., Pool, J. E., Kassner, V. A., Aquadro, C. F., Carroll, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stepwise+modification+of+a+modular+enhancer+underlies+adaptation+in+a+Drosophila+population.">Stepwise modification of a modular enhancer underlies adaptation in a Drosophila population.</a> Science. 326, 1663-1667 (2009).</li><li>Williams, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+and+evolution+of+a+genetic+switch+controlling+sexually+dimorphic+traits+in+Drosophila.">The regulation and evolution of a genetic switch controlling sexually dimorphic traits in Drosophila.</a> Cell. 134, 610-623 (2008).</li><li>Shirangi, T. R., Dufour, H. D., Williams, T. M., Carroll, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+evolution+of+sex+pheromone-producing+enzyme+expression+in+Drosophila.">Rapid evolution of sex pheromone-producing enzyme expression in Drosophila.</a> PLoS biology. 7, e1000168-e1000168 (2009).</li><li>Barolo, S., Carver, L. A., Posakony, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFP+and+beta-galactosidase+transformation+vectors+for+promoter/enhancer+analysis+in+Drosophila.">GFP and beta-galactosidase transformation vectors for promoter/enhancer analysis in Drosophila.</a> BioTechniques. 29, 726-732 (2000).</li><li>Fish, M. P., Groth, A. C., Calos, M. P., Nusse, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Creating transgenic Drosophila by microinjecting the site-specific phiC31 integrase mRNA and a transgene-containing donor plasmid. , (2007).</li><li>Ashburner, M., Golic, K. G., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila: A laboratory handbook. , (2005).</li><li>Abramoff, M. D., Magelhaes, P. S., Ram, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+Processing+with+ImageJ.">Image Processing with ImageJ.</a> Biophotonics International. 11, 36-42 (2004).</li><li>Musunuru, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+noncoding+variant+to+phenotype+via+SORT1+at+the+1p13+cholesterol+locus.">From noncoding variant to phenotype via SORT1 at the 1p13 cholesterol locus.</a> Nature. 466, 714-721 (2010).</li></ol>

cis-regulatory elements encode the genomic program that specifies gene expression patterns and thereby the process of development1, and are prominent locations for both mutations underlying morphological evolution2-4 and phenotypic variation for human traits5,6,19. In spite of this importance, the regulatory logic for CREs remain poorly understood. A prominent reason for this understanding deficit has been the lack of suitable methods to quantitatively compare functional sequence...

quantitative comparison of cis-regulatory element (cre) activities in transgenic drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Watch this Scientific Journal Video about Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster at JoVE.com

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

quantitative-comparison-cis-regulatory-element-cre-activities

Research

JoVE Journal

Biology

11.7K ビュー.  University of Dayton. 形質の表現型変異は、シス調節エレメント（CRE）のシーケンスを制御する遺伝子の発現パターンの変異に起因することができます。キイロショウジョウバエで使用するために派生メソッドは、定量的に変更されたり、天然に存在するCREの亜種によって媒介遺伝子発現の空間的および時間的パターンのレベルを比較することができます。