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Paper text:
the uncharacterized gal4 enhancer trap lines screened in this study were obtained from the getdb , a subdivision of the drosophila genetic resource center , kyoto institute of technology , japan .
the following lines were obtained from the bloomington drosophila stock center : hh - lacz , ptc - gal4 , ptc - lacz , dpp - gal4 , dpp - lacz , and ok107-gal4 .
other drivers used in this study : antp - gal4 ( s. cohen , temasek life sciences laboratory , singapore ) , cg - gal4 ( c. dearlolf , massachusetts general hospital , boston ) , dome - gal4 ( s. noselli , centre national de la recherche scientifique , nice , france ) , dot - gal4 ( d. kimbrell , university of california , davis ) , gcm - gal4 ( l. zipursky , university of california , los angeles ) , hand - gal4 ( z. han , university of michigan ) , hh - gal4 ( t. kornberg , university of california , san francisco ) , upd - gal4 ( y.h . sun , academia sinica , taiwan ) .
approximately 2 kb of dna immediately upstream of the ubiquitin - p63e ( cg11624 ) open reading frame was pcr amplified as an ecori / kpni fragment from a previously cloned promoter region ( kindly provided by p. o'farrell , university of california , san francisco ) and cloned into the pcrii vector ( invitrogen ) .
this ecori / kpni fragment was then subcloned into the drosophila transformation vector pstinger ( drosophila genomics resource center ) , which contains a multiple cloning site upstream of the nuclear egfp reading frame
, containing a transcriptional termination sequence flanked by an inverted pair of frt recombination sites , was isolated as a kpni fragment from the construct d237 ( kindly provided by g. struhl , columbia university ) and subcloned into the kpni site that lies in between the ubi - p63e promoter and the egfp reading frame .
transgenic flies were generated using the pubi-63e - stop - stinger vector and a commercial transformation service ( bestgene , inc . ) .
a triple - recombinant chromosome was generated using uas - redstinger14 ( w ; p{uas - redstinger}4/cyo , bloomington drosophila stock center ) , uas - flp ( yw * ; p{uas - flp1.d}jd1 , bloomington drosophila stock center ) and pubi-63e - stop - stinger insert 9f6 and was balanced over cyo .
the gal4-expressing lines were individually crossed on standard fly food at 25c . wandering third instar larvae
were dissected in 1xpbs and tissues were fixed in 4% formaldehyde in 1xpbs for 30 min , followed by three washes ( 10 min each ) with 1xpbs/0.4% triton - x-100 ( fisher ) detergent ( pbst ) . during the second 1xpbst wash , to - pro3 iodide ( invitrogen )
tissues were mounted on slides in vectashield ( vector laboratories ) and imaged using a zeiss apotome or a biorad radiance 2000 confocal microscope with standard techniques .
the uncharacterized gal4 enhancer trap lines screened in this study were obtained from the getdb , a subdivision of the drosophila genetic resource center , kyoto institute of technology , japan .
the following lines were obtained from the bloomington drosophila stock center : hh - lacz , ptc - gal4 , ptc - lacz , dpp - gal4 , dpp - lacz , and ok107-gal4 .
other drivers used in this study : antp - gal4 ( s. cohen , temasek life sciences laboratory , singapore ) , cg - gal4 ( c. dearlolf , massachusetts general hospital , boston ) , dome - gal4 ( s. noselli , centre national de la recherche scientifique , nice , france ) , dot - gal4 ( d. kimbrell , university of california , davis ) , gcm - gal4 ( l. zipursky , university of california , los angeles ) , hand - gal4 ( z. han , university of michigan ) , hh - gal4 ( t. kornberg , university of california , san francisco ) , upd - gal4 ( y.h . sun , academia sinica , taiwan ) .
approximately 2 kb of dna immediately upstream of the ubiquitin - p63e ( cg11624 ) open reading frame was pcr amplified as an ecori / kpni fragment from a previously cloned promoter region ( kindly provided by p. o'farrell , university of california , san francisco ) and cloned into the pcrii vector ( invitrogen ) .
this ecori / kpni fragment was then subcloned into the drosophila transformation vector pstinger ( drosophila genomics resource center ) , which contains a multiple cloning site upstream of the nuclear egfp reading frame .
, containing a transcriptional termination sequence flanked by an inverted pair of frt recombination sites , was isolated as a kpni fragment from the construct d237 ( kindly provided by g. struhl , columbia university ) and subcloned into the kpni site that lies in between the ubi - p63e promoter and the egfp reading frame .
transgenic flies were generated using the pubi-63e - stop - stinger vector and a commercial transformation service ( bestgene , inc . ) .
a triple - recombinant chromosome was generated using uas - redstinger14 ( w ; p{uas - redstinger}4/cyo , bloomington drosophila stock center ) , uas - flp ( yw * ; p{uas - flp1.d}jd1 , bloomington drosophila stock center ) and pubi-63e - stop - stinger insert 9f6 and was balanced over cyo .
the gal4-expressing lines were individually crossed on standard fly food at 25c . wandering third instar larvae
were dissected in 1xpbs and tissues were fixed in 4% formaldehyde in 1xpbs for 30 min , followed by three washes ( 10 min each ) with 1xpbs/0.4% triton - x-100 ( fisher ) detergent ( pbst ) . during the second 1xpbst wash , to - pro3 iodide ( invitrogen )
tissues were mounted on slides in vectashield ( vector laboratories ) and imaged using a zeiss apotome or a biorad radiance 2000 confocal microscope with standard techniques .
we combine gal4/uas , flp / frt and fluorescent reporters to generate cell clones that provide spatial , temporal , and genetic information about the origins of individual cells in drosophila . 
 we name this combination the gal4 technique for real - time and clonal expression ( g - trace ) . 
 the approach should allow for screening and the identification of real - time and lineage - traced expression patterns on a genomic scale .