Drosophila Reporter Vectors Compatible with ?C31 Integrase Transgenesis Techniques and Their Use to Generate New Notch Reporter Fly Lines.

1.1 MB·English

by  Housden, Ben E.

INVESTIGATION
Drosophila
Reporter Vectors Compatible with
F
C31 Integrase Transgenesis Techniques and Their
Use to Generate New Notch Reporter Fly Lines
Ben E.Housden,KatMillen, andSarahJ.Bray1
DepartmentofPhysiology,DevelopmentandNeuroscience,UniversityofCambridge,CambridgeCB23DY,United
Kingdom
ABSTRACT Complexspatialandtemporalregulationofgeneactivityisfundamentaltodevelopmentand KEYWORDS
homeostasis. The ability to decipher the DNA sequences that accurately coordinate gene expression is, reporterplasmids
therefore,ofprimaryimportance.OnewaytoassessthefunctionsofDNAelementsentailstheirfusionto FC31integrase
fluorescent reporter genes. This powerful approach makes it possible to visualize their regulatory fluorescent
capabilitieswhenreintroducedintothedevelopinganimal.TransgenicstudiesinDrosophilahaverecently protein
advanced with the introduction of site-specific, FC31 integrase–mediated approaches. However, most Drosophila
existingDrosophilareportervectorsarenotcompatiblewiththisnewapproachandhavebecomeobsolete. Notch
HerewedescribeanewseriesoffluorescentreportervectorsoptimizedforusewithFC31transgenesis.By
usingthesevectorstogenerateasetofNotchreporterflylines,wedemonstratetheirefficacyinreporting
thefunctionofgeneregulatoryelements.
UnderstandingthemechanismsemployedbyregulatoryDNAsequen- More recently, a transformation system has been introduced
cestocontrolgeneexpressionisessentialtothestudyofdevelopment which exploits the integration mechanism used by bacteriophage
and disease.Tools enabling this are, therefore, ofmajor importance. FC31(Grothetal.2004).Aphageintegraseinducesrecombination
Onemethodfordecipheringgenomicregulatoryinformationutilizes betweenattP(phagegenome)andattB(bacterialgenome)sequences
invivoreporterassaystoassesstheactivityofputativeenhancers.For (Groth and Calos 2004; Thorpe and Smith 1998). Several groups
such analysis, the DNA is subcloned adjacent to easily monitored haveestablishedtransgenicflylinescontainingattPsites(platforms)
reportergenes,suchasencodingb-galactosidaseorgreenfluorescent at specific, nonmutagenic locations (Bateman et al. 2006; Bischof
protein(GFP),invectorsdesignedfortransgenesis.Thisapproachhas et al. 2007; Groth et al. 2004; Markstein et al. 2008; Venken et al.
been widely exploited in Drosophila, where stably inherited, single- 2006). Injection of attB-containing vectors with a source of FC31
copy transgene insertions could be generated with p-element trans- integraseresultsinintegrationofthevectorintothegenomeatthe
poson-basedvectors(RubinandSpradling1982;SpradlingandRubin attPplatform.
1982). With this method, transgenes are inserted randomly in the TheFC31systemismoreefficientthanprevioustechniques,and
genome, withapreferenceforpromoter regions(Bellen etal.2004). as integration occurs at specific attP sites, insertions are directly
Expressionfromsuchinsertionsisofteninfluencedbythesurround- comparable and mapping is unnecessary. However, most vectors
ingsequences (positioneffects),leadingtodifficultiesininterpreting forinvivoreporterassays,lackingattBsites,areincompatiblewith
patternsgenerated. thismethod.TwoadaptedvectorswithattBsequenceshaverecently
beenmade,butbothuseGatewaycloningandoneretainsp-element
ends, precluding subsequent use of p-element mutagenesis in the
Copyright©2012Housdenetal. fliesgenerated(Aertsetal.2010;Boyetal.2010).Wehavegenerated
doi:10.1534/g3.111.001321
a new series of compatible vectors that contain no unneccessary
ManuscriptreceivedAugust18,2011;acceptedforpublicationNovember9,2011
This is an open-access article distributed under the terms of the Creative sequences and are optimized for enhancer detection due to the
CommonsAttributionUnportedLicense(http://creativecommons.org/licenses/ positionofthecloningsite,inclusionofinsulators,anduseofmul-
by/3.0/),whichpermitsunrestricteduse,distribution,andreproductioninany tiple reporters.
medium,providedtheoriginalworkisproperlycited.
To achieve this,weadaptedelementsfromtheexistingHZ50PL-
1Correspondingauthor:DepartmentofPhysiology,Development,and
lacZenhancer–detectingvector(HiromiandGehring1987)andahigh
Neuroscience,UniversityofCambridge,DowningStreet,CambridgeCB23DY,
UK. E-mail:[email protected] copy p-element transformation plasmid [p-WhiteRabbit; Dunin-
Volume2 | January 2012 | 79
Borkowski and Brown (1995)]. We combined the hsp70 minimal Vectorsaccurately reportNotchresponsive
promoter from HZ50PL-lacZ with eGFP, mCherry, lacZ, or venus enhanceractivity
[PEST]-YFP coding sequences. Incorporating these reporters into To test the functionality of these vectors, we inserted a previously
a plasmid containing the p-WhiteRabbit vector backbone, mini- characterized Notch responsive element (NRE) (Furriols and Bray
white, and kanamycin resistance genes (kan) in combination with 2001). Costaining to detect expression from NRE-GreenRabbit and
anattBsequenceenablesuseoftheFC31system.Alox-psitewas the previous NRE-lacZ (Figure 2, B-B”) revealed identical patterns
included to allow removal of kan and platform sequences after in wing discs. Furthermore, NRE-GreenRabbitgavethe expected ex-
genomicintegration. pression patterns elsewhere. In trachea, GFP was detected only in
Tominimizeinfluencefrompositioneffects,themini-whitegene nestsofcellsatthetrachealbranchpoints,asreportedfortheparent
and vector backbone are arranged to flank the reporter gene after NRE reporter (Furriols and Bray 2001), and in larval brains, it was
integration (Figure 1). We also inserted insulator (gypsy) elements, present in imaginal neuroblasts, a known site of Notch activity
which have been shown to be effective in reducing the influence of (Almeida and Bray 2005) (Figure 2, I and J). Notch responsiveness
neighboring sequences, flanking mini-white and the reporter gene of NRE-GreenRabbit was confirmed by expressing Notch-RNAi in
(Barolo et al. 2000; Barolo et al. 2004) (Figure 1, purple circles). posterior compartments of wing discs. Under these conditions, ex-
Resulting vectors are named after the originating plasmid pression waslost, indicatingthatit is dependenton Notch signaling
(pWhiteRabbit), substituting the color prefix according to the type (Figure2F).NREfunctionwasalsoaccuratelyreportedwithmCherry,
ofreporter (pGreenRabbit, etc.). Thesevectors arecompatible with lacZ, and Venus[PEST]-YFP variants (Figure 2, C-E). As expected,
a wide range of experiments, including live imaging. For example, independent insertions of NRE-RedRabbit at a single platform site
destabilized Venus[PEST]-YFP could be used when perdurance of gave reproducible expression levels compared with independent
the reporter would be an issue or when fine-scale temporal differ- NRE-lacZ p-element insertions (Figure 2K). The new vectors, there-
encesinexpressionareinvestigated(Aulehlaetal.2008;Nagaietal. fore, accurately and reproducibly report known expression patterns
2002).Furthermore,thedifferentreportersenableseveralregulatory fromenhancerelements.Thelinesgeneratedwillalsoprovideuseful
elementstobeanalyzed simultaneously. toolsforanalysisofNotchpathwayactivityinvivo.
Reportersshowno basalexpression Transgenesareresistant topositioneffects
One important criterion for reporter constructs is that basal Totestsusceptibilitytosurroundingsequences,weanalyzedexpression
expression levels should be low. We tested whether pGreenRabbit from pGreenRabbit at a location prone to position effects from a
gave any expression in the absence of an enhancer by generating neighboring gene (51D) (http://flyc31.frontiers-in-genetics.org/). No ex-
insertionsinseveralattPplatformlines(2A,22A,51D,68E,81C,and pression was detected in wing imaginal discs (Figure 3A) or in several
96E).InnocasewasGFPexpressiondetectedinthewingdisc(Figure other tissues (Figure 2, G and H), indicating that the flanking vector
2A and data not shown). Similarly, no basal expression could be sequencesareeffectivebuffers.Whenthesevectorswereremovedbyin-
detected in larval brains or trachea, confirming their efficacy as en- ducingrecombinationbetweenlox-psites,patternedreporterexpression
hancer-detectionvectors(Figure2,GandH). waspresentbutgreatlyattenuatedinthepresenceofinsulators(Figure3,
Figure1 Reportervectorscom-
patiblewithFC31transgenesis
techniques. Diagram of vector
backbone (top) into which four
different reporters have been
inserted as indicated. All vec-
tors carry kanamycin resistance
andusemini-whiteasatransfor-
mationmarker.Allreportersare
downstreamofaminimalhsp70
promoter and are flanked by
UTRs from the hsp70 gene.
The positions of gypsy (SuHw)
insulator sequences (purple),
lox-p, attB (yellow), and multi-
ple cloning site (MCS) unique
restrictionsitesareindicated.
80 | B.Housden,K.Millen,andS.J.Bray
Figure2 VectorsaccuratelyreportexpressionpatternfromaNotchresponsiveelement.(A)BasalexpressionfrompGreenRabbit(GR)integrated
atplatform51D.(B–E)ExpressionfromindicatedreportervectorsdrivenbytheNotchresponsiveelement(NRE)inthewingpouchofthirdinstar
larvae.NRE-lacZ(B,magenta;B99,white)isapreviouslyreportedNotchreporter(FurriolsandBray2001).ThesameNREwasusedtogenerate
NRE-GreenRabbit(NRE-GRins;B,green;B9,white);NRE-RedRabbit(NRE-RRins;C);NRE-BlueRabbit(NRE-BRins;D);andNRE-VenusRabbit(NRE-
VRins;E).NRE-VRinswasimagedwith10·excitation.(F)Notch-RNAiexpressionintheposteriorcompartment(greenbraces;drivenusingen-
Gal4)eliminatesexpressionfromNRE-GRins.(G,H)BasalexpressionfrompGRintegratedatplatform51Dinthelarvalbrain(G)andtrachea(H).(I,
J)ExpressionfromNRE-GRinsinthelarvalbrain(I)andtrachea(J).Suffix“ins”indicatesthattheconstructscontainedinsulators.(K)Comparisonof
expressionlevelsfromindependenttransgeniclines(averagepixelintensityinthewingpouchmeasuredusingImageJ).NRE-RRinsertionsatthe
sameplatform(86Fb)producesimilarexpressionlevels.NRE-lacZinsertionsgeneratedusingp-elementtransgenesisareexpressedatvarying
levels. At least five discs were quantified per genotype. Error bars show standard error of the mean. Primary antibodies were rabbit a-GFP
(MolecularProbes,1/500)(A,B,andE–J);rabbita-dsRed(ClonTech,1/50)(C);andmousea-bGalactosidase(DevelopmentalStudiesHybridoma
Bank,1/20)(BandD).
B-C).Therefore,boththebufferingsequencesandinsulatorsareeffective Insummary,wehaveconstructedaseriesoffourreportervectors
inpreventingpositioneffectsandmakethevectorsresistanttoinfluences specifically designed for use with FC31-mediated transgenesis that
fromsurroundingDNA.Furthermore,theinsulatorsequenceshaveno enable analysis and direct comparisons of different enhancers. Insu-
adverseeffectsonvectorfunction,(Figure3,DandE). lator elements and buffering sequences have been incorporated to
Volume2 January2012 | FC31-CompatibleReporterVectors | 81
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