for our analysis of rab gtpases , we exclusively used a gal4 targeting cassette ( figs .  1 , 2a ) .
here we provide four additional cassettes : ( 1 ) a simple 3xp3 rfp/ kan cassette for making molecularly defined deletions and ( 2 ) hemagglutinin ( ha ) , ( 3 ) flag and ( 4 ) green fluorescent protein ( gfp ) cassettes for tagging gene products ( fig
these cassettes , along with the gal4 cassette , all have the 3xp3 rfp / kan sequences flanked by loxp sites , which allows for positive selection in both bacteria and in flies and has proved to be invaluable for a number of different reasons ( see below ) .
moreover , this cassette can be removed in vivo using available cre transgenic lines ( bloomington stock center : bl#1501 ) .
the activity of cre leaves behind a 34 bp scar that should be taken into account if one attempts to tag gene products at their n - termini .
the gfp , ha and flag tagging cassettes share common 5 and 3 sequences , allowing one set of locus specific primers to be used across the entire cassette collection ( fig . 
( b ) schematic showing the specific sequence flanking each of the specific tagging cassettes .
details on recombineering methods have been presented elsewhere . briefly , our protocol follows four basic steps for vector building ( fig . 
1 ) : ( 1 ) pcr amplify and pcr - soe 500 bp left arm ( la ) and right arm ( ra ) homology arms that flank both ends of the genomic region of interest together and clone them into the frt / i - sce i modified p[acman]-ko 1.0 vector .
( 2 ) use gap repair within recombination competent bacteria ( dy380 ) to clone the entire region of interest into p[acman]-ko 1.0 .
( 3 ) amplify the targeting cassette using oligos containing 100 bp of homology to either side of the gene being targeted . ( 4 ) transform dy380 cells carrying the p[acman]-ko - region of interest - plasmid with the pcr amplified targeting cassette .
we simplified the first recombineering step by incorporating gateway cloning sequence into our modified p[acman]-ko vector .
when designing primers , 25 bp of attb sequence is included on the 5 end of the forward la primer and the reverse ra primer . including these attb sequences on either end of the pcr - soe product
allows one to clone this dna into p[acman]-ko using a bp reaction ( invitrogen ) .
incomplete digestion of the p[acman]-ko- pcr - soe product plasmid used for gap repair in the first recombineering reaction ( fig .  1 ;
therefore we use high - fidelity enzymes and allow the digestion of the p[acman]-ko- pcr - soe product to continue for at least three hours .
while the number is variable from construct to construct , we typically obtain < 50 amp / tet resistant dy380 colonies after the first recombineering reaction . a large number of amp / tet resistant colonies after the gap repair reaction usually indicates the presence of the original unrecombineered p[acman]-ko pcr - soe product plasmid . when creating a targeting cassette specific for a particular gene , it is imperative to amplify from a completely linearized and purified cassette template . attempting to pcr directly off the plasmid carrying a particular cassette
kanamycin resistance conveyed by a contaminating plasmid results in a high number of false positive dy380 colonies after the second recombineering reaction ( fig .  1 ; step 4 ) .
because of this observation , we often run dna from colonies obtained after step 4 on an agarose gel to check for the presence of a low molecular weight high copy plasmid before transforming the final vector into epi300 cells . after sequence verification , correctly recombineered plasmids are transformed into epi300 cells , amplified using copy control solution ( epicenter ) and  maxi- prepped for injection as outlined in chan et al . ( 2011 ) .
importantly , we found that freshly prepared dna works best for transformation and therefore always performed the maxi - prep not more than one day before injection .
moreover , the large molecular weight dna should be stored at 4c and never frozen . despite these steps we still find that transformation efficiencies tend to be low for p[acman ] vectors over 50 kb ( table 1 ) . because of this low efficiency , we maximize the number of progeny obtained of every g0 fly .
single g0 males were crossed to 35 virgin females and single g0 females were crossed to 3 males and all these vials are tossed up to three times .
the dna corresponding to rab5 , rab40 , rabx1 , rab2-atg and rab18 gal4 vectors was not diluted prior to transformation into epi300 cells , resulting in drosophila transformants that did not carry the gal4 targeting cassette . diluting the dna of remaining rabs prior to introducing the plasmid into epi300 cells alleviated this problem so that all the subsequent drosophila transformants carried the gal4 cassette .
mobilization of the targeting cassette from the predefined insertion site in vivo is performed using existing transgenic sources of flpase and i - sce i ( bloomington stock # s : 6934 and 6935 ) .
this mobilization results in the targeting of the knock - in / out cassette to the endogenous locus .
p[acman]-ko transformants are identified based on the presence of the white+ gene contained within the backbone of the p[acman]-ko 1.0 vector . through the course of our experiments we sometimes isolated transformed flies that expressed the white+ marker but did not carry the 3xp3-rfp gene contained within the gal4 targeting cassette ( table 1 ) .
furthermore , we isolated white+ , rfp+ and white+ , rfp- flies from the same g0 parent .
these observations suggested that the dna used for transformation contained a mixture of p[acman]-ko plasmids : some that contained the gal4 3xp3-rfp cassette and others that did not .
we considered the possibility that the p[acman]-ko vector was kept at a low copy number in dy380 cells but not at single copy .
therefore it remained possible that the gal4 cassette inserted in some fraction of the p[acman ] plasmids within a particular cell but not all of them .
if dna isolated after the final recombineering reaction is indeed a mixture , simply diluting the dna before transforming epi300 cells should greatly reduce the probability that any resulting bacterial colony would contain both types of plasmids .
subsequent experiments showed that diluting dna to the point where one obtains 150 epi300 amp / kan resistant colonies on a 35 mm petri dish after electroporation virtually eliminates false positive ( white+ , rfp- ) drosophila transformants .
therefore inclusion of the positive rfp+ marker within the targeting and tagging cassettes provides insurance that drosophila transformants carry correctly recombineered vectors .
the key step in a homologous recombination experiment in drosophila ( and the main difference to mouse ) is the mobilization of the targeting cassette from a defined site where it is already integrated in the genome .
hence , instead of injection into embryonic stem ( es ) cells , the targeting cassette is mobilized by enzymatic excision in germ cells .
consequently , selection screening for a correct targeting event , i.e. , insertion of the targeting cassette into the correct endogenous locus , has to occur in flies instead of cells .
this process requires three steps : first , the donor dna ( targeting cassette ) and transgenes encoding the enzymes needed to mobilize the cassette ( hs - flp and hs - i - sce i ) are crossed into the same genetic background .
second , preliminary candidates are screened for mobilization of the targeting cassette away from its original site . this step is greatly facilitated if both the targeting cassette and the site from which it is mobilized are independently marked .
indeed , this feature constitutes a major difference between our technique and other recent efforts , as outlined in detail below .
the third step comprises mapping new insertions of the targeting cassette to the correct chromosome , followed by molecular and/or genetic verification .
it is important to note that most insertions are not the desired clean homologous recombination event .
several modifications have been introduced to optimize this process , which we would like to compare with our recently developed method .
major improvements to ends - out homologous recombination have recently been introduced by huang et al .
first , a dominant and heat shock dependent cell lethal construct ( hs - hid ) present on both the y and balancer chromosomes kill off all undesired progeny .
second , a dominant cell lethal construct ( uas - reaper ) is fused behind the 3 homology arm of the targeting cassette . in a correct targeting event
, this dna fragment is deleted ; however , in a faulty targeting event its presence leads to the death of the undesired offspring when driven by a neuronal or ubiquitous gal4 driver .
the latter improvement ( uas - reaper ) has so far not been tested in conjunction with our technique , and a combined method might substantially improve targeting efficiency . a further major difference between our approach and the method by huang et al .
use white+ as a positive selection marker of the targeting cassette but do not mark the site from which it is mobilized , leading to a difficulty in distinguishing failed mobilization or mobilization and re - insertion based on white+ alone . in theory ,
offspring in which the targeting cassette fails to mobilize should be killed by the uas - reaper dominant negative selection .
hence , all viable and white+ progeny should represent cases where the targeting cassette was mobilized and re - integrated without the uas - reaper sequence .
since random re - insertion is predicted to result in a higher rate of insertion on the correct chromosome , the majority of these false positive 
in contrast , our technique employs positive labeling of the targeting cassette with 3xp3-rfp and white+ , yellow+ as independent markers for the site from which the targeting cassette is mobilized .
selection for the targeting cassette ( rfp+ ) and against the site from which it is mobilized ( white- and yellow- ) significantly reduces the number of preliminary candidates .
report between 116 and 1700 preliminary candidates of which between 1 and 22 could be verified as correct targeting events .
in contrast , our method leads to 20100 preliminary candidates of which 8090% are on the correct targeted chromosome .
the accuracy of these targeting events depends on the homology region and was reported for one locus in chan et al .
the recombineering methods outlined here and in our recent study provide a means to manipulate the drosophila genome with base - pair precision .
recombineering represents a powerful and scalable method for determining gene function and for exploring how locus dependent and independent parameters contribute to the efficiency of in vivo homologous recombination in drosophila .
 evaluating how an individual gene contributes to a particular biological process benefits greatly from a comprehensive understanding of all members of its gene family . 
 such knowledge is ideally obtained using multicellular model organisms , which provide rapid and decisive platforms for determining gene function . 
 we recently established a novel transgenesis platform in drosophila to systematically knock out all members of the rab small gtpase family of membrane regulators . 
 this platform combines bac transgenesis / recombineering with ends - out homologous recombinations and gatewaytm technologies and provides a new rapid and scalable method that eases the manipulation of endogenous loci . 
 this method not only allows for the generation of molecularly defined lesions , but also the precise replacement or tagging of genes in their endogenous loci . using this method 
 , we found that up to half of all rab gtpases exhibit enriched expression at synapses in the nervous system . 
 here we provide critical details about the underlying recombineering and transgenesis method , new cassettes for tagging endogenous loci and information on important parameters that will allow drosophila researchers to target members of other gene families . 
New Replacement and Tagging Cassettes for Recombineering
Methodological Considerations for Recombineering and Transformation
Gene Targeting of Endogenous Loci
Closing Remarks