BACKGROUND
the generation of transgenic animals in model systems such as caenorhabditis elegans has created dramatic changes in the ability of researchers to approach biologic questions  <cit> .
it is relatively straightforward to create and use a transgenic animal to investigate the timing and pattern of gene expression, express epitope-tagged versions of genes, or test the effects of gene mis-expression.
traditionally, constructs used for creation of a gfp fusion gene or epitope-tagged version of a gene were drawn from regions of cloned genomic dna which were known to rescue mutants of the specific gene  <cit> .
as the availability of rnai, microarrays, and other approaches which are based upon the worm genomic sequence instead of classical mutants has increased, a need to create transgenes in other ways has developed.
for these genes, a region 5' of the gene is often isolated via pcr and then used as a promoter  <cit> .
this approach has been further simplified by the construction of a large number of vectors by the fire lab and later by the generation of the c. elegans promoterome  <cit> .
however, the decision about the exact length and location of the promoter is more often driven by arbitrary decisions about the size  or convenient restriction sites than sequence analysis.
as a result, it is difficult to know if the gfp expression pattern created by a selected promoter reflects the true expression pattern of the gene in vivo  <cit> .
to generate transgenes that are more likely to reflect the in vivo expression timing and pattern, interest in generating transgenics using the larger regions of genomic dna carried by either fosmids or bacs  is growing  <cit> .
this approach carries the advantage of using 30– <dig> kb of genomic dna in the construction of a transgene so it is likely that all or most of the promoter and enhancer elements are included.
additionally since the genomic dna for the gene is included, additional complexities such as alternative splicing, multiple transcription start sites, and regulation by 3' utr sequences or microrna can also occur.
for this approach to become a standard technique within the c. elegans community, approaches need to be developed to make the modification of fosmids and bacs quick and robust for the novice.
two recent publications have begun to lay the groundwork for this to occur  <cit> .
the first described the modification of c. elegans fosmids using homologous recombination in e. coli  using pcr generated dna fragments  <cit> .
the creation of a transgene occurred through a two-step procedure where first a teta-rpsl cassette  flanked by homology arms is inserted at the desired location and then in a second step the cassette is replaced by the desired dna, such as gfp, flanked by the same homology arms.
this technique allows the insertion of essentially any sequence at any site in the gene.
the second paper described the modification of bacs from the related nematode species caenorhabditis briggsae also by the use of recombineering  <cit> .
in this protocol, a gfp fusion gene is created by inserting gfp at the c-terminus of a gene of interest along with a flanking kanamycin resistance cassette via a single round of recombineering with a pcr product flanked by homology arms.
the bac is also prepared for use as a transgene by insertion of the unc- <dig> gene via recombineering.
the addition of the unc- <dig> gene provides an easily visualized selectable marker as this dna is able to rescue the size, body shape, mobility, and starvation resistance defects present in unc- <dig> mutants  <cit> .
each approach has strengths and weaknesses for the routine generation of transgenic worms  <cit> .
we have furthered their work by developing a streamlined series of procedures and accompanying vectors to allow the simple and reliable generation of transgenes ready for the transformation of worms by bombardment or microinjection.
specifically, we focused on the use of c. elegans genomic dna cloned into a commercially available fosmid library .
we then developed tools that will allow the robust and efficient generation of either flag-gfp or tap fusion genes by four important changes.
first, we utilized the more robust galk gene for positive and then negative selection during recombineering  <cit> .
second, we constructed plasmids for galk utilizing the r6k origin which is unable to replicate in the e. coli strain used for recombineering and minimizes the amount of processing of the pcr products used  <cit> .
third, we built homology arms for both gfp and n and c-terminal tap tags into the galk cassette so that a single pair of oligos can be used for the generation of pcr products for each step.
the oligos used for recombineering are long and require purification prior to use so the need for one pair instead of two reduces either the labor or cost involved in procuring the oligos.
finally, we developed a protocol for adding the unc- <dig> gene to the fosmid backbone through cre-lox mediated recombination  <cit> .
the final fosmid product can then be used directly for the generation of transgenic nematodes.
RESULTS
we sought to use the c. elegans fosmid library and recombineering for the routine generation of transgenes and subsequent creation of transgenic worms.
two techniques have been described using recombineering in c. elegans with one using a bac library generated from the related nematode species c. briggsae while the other used the fosmid library followed by microinjection of the modified plasmids  <cit> .
we initially used the protocol described by dolphin et.
al.
to successfully generate several daf- <dig> transgenes  .
during this process we identified several ways to optimize and simplify the protocol for recombineering and transgenic animal creation.
the plasmid template used for the generation of the selection/counter-selection cassette by pcr, pbac-rt, is able to replicate in the sw <dig> bacterial strain used for recombineering.
consequently, this template needs to be destroyed prior to electroporation either by dpni digestion or as suggested by dolphin et.
al.
through the use of a restriction fragment as the pcr template  <cit> .
as pbac-rt is constructed on a bac vector backbone, it is a very low copy number plasmid, and we experienced difficulty in purifying sufficient dna to digest and gel purify the fragment containing the rt cassette.
to address this difficulty, we subcloned the rt cassette from pbac-rt into the pmod <dig> vector .
pmod <dig> uses the r6k replication origin which requires the π protein for replication in e. coli.
the resulting plasmid pmod <dig> rt has the advantages of having a higher copy number than pbac-rt which simplifies dna purification and of being unable to replicate in the sw <dig> bacterial strain.
even when > <dig> ng.
of pmod <dig> rt dna was electroporated into sw <dig> no tetr colonies were obtained .
as a result, pcr products used for recombineering do not need to be digested with dpni or be extensively purified prior to use.
for c. elegans experiments most transgenes consist of gfp fusions constructed to either identify the location and timing of gene expression and/or to visualize the cellular localization of the protein.
to construct a gfp fusion transgene using the method of dolphin et.
al.
two pairs of long oligonucleotides need to be ordered and purified for the generation of a transgene  <cit> .
the first set anneal to the rt cassette and carry  <dig> base pair homology arms specific for the gene of interest.
the second set anneals to gfp and carry the same homology arms.
if these are purchased from commercial companies the cost of these oligos is not insignificant especially if multiple transgenes are to be generated.
to facilitate the construction of gfp fusion genes, we inserted oligos with  <dig> base pair homology arms for gfp into pmod <dig> rt to generate pmod <dig> rt-g .
the presence of these homology arms allows a single set of oligos to be used for the amplification of the rt cassette and gfp .
this reduces the cost for oligos by half compared with the prior technique.
during the construction of pmod <dig> rt and daf- <dig> transgenes using the rt cassette we found two limitations of the rt cassette.
first, the rt cassette appears to be somewhat toxic to e. coli as pmod <dig> rt could only be maintained in the jm109λpir strain where it is a low copy number plasmid instead of the ec100pir- <dig> strain which increases the copy number of r6k based plasmids to ~ <dig>  this toxicity may contribute to some of the difficulties we experienced with positive and negative selection of fosmids carrying the cassette.
second, the rt cassette involves separate genes for positive and negative selection during recombineering  <cit> .
this property may increase the background during negative selection as mutations in the gene involved would be silent until the bacteria are plated on selective media  <cit> .
these mutations are most likely introduced during pcr amplification as  <dig> amplification of the template would be expected to produce ~2–6% of products with mutations for proofreading polymerases and up to 25% for taq-proofreading polymerase blends  <cit> .
hence, any toxicity of the cassette would facilitate selection of mutated, less toxic clones.
to address these two concerns we experimented with the use of the galk gene for positive and negative selection during recombineering  <cit> .
to facilitate the use of galk, we transferred the galk gene to the pmod <dig> plasmid and added gfp homology arms for use during recombineering .
we initially compared the use of pmod <dig> rt-g and pmod <dig> galk-g by constructing a gfp fusion at the n-term of the k10c <dig>  gene which encodes a member of the fumarylacetoacetate hydrolase family   <cit> .
prior attempts to create a gfp reporter using the promoterome library clone were unsuccessful perhaps due to a lack of important promoter elements in the promoter clone .
we found that the rt and galk cassettes can be easily inserted into a fosmid carrying k10c <dig>  by recombineering .
we then used a pcr fragment carrying flag-tagged gfp to replace either the rt or galk cassette via a second round of recombineering.
colonies with successful substitution of flag-gfp were identified by using selective media.
we found the galk cassette was more robust than the rt cassette as 4/ <dig> colonies created with the galk cassette and 0/ <dig> colonies created with the rt cassette  carried flag-gfp by pcr.
pcr analysis of a further  <dig> clones revealed 40/ <dig> colonies created with the galk cassette and 0/ <dig> colonies created with the rt cassette carried flag-gfp after selection .
dod-22
myo-3
the source of this difference appears to be at least in part due to weaker negative selection from the rt cassette as revealed by analysis of colonies obtained after a no pcr product control electroporation.
this control was conducted similarly to the second recombineering step but no flag-gfp pcr product was added prior to electroporation.
pcr analysis of  <dig> colonies for the galk and rt cassettes revealed that 23/ <dig> galk cassette bacteria still had at least part of the cassette whereas 46/ <dig> rt colonies still had a cassette detectable by pcr .
this suggests that a significant number of bacteria with the rt cassette are still able to grow despite negative selection.
further, addition of the flag-gfp pcr product to the electroporation resulted in a noticeable increase in colonies from bacteria with the galk cassette but not from bacteria with the rt cassette .
these counts were performed in parallel and included bacteria from undiluted, 1: <dig> diluted, and 1: <dig> diluted samples plated from a single experiment.
together, the greater number of total colonies obtained from bacteria with the galk cassette along with the more effective negative selection might lead to the greater effectiveness of the galk cassette in recombineering as the significant number of true positives could now outnumber the relatively small number of false positives.
consistent with this, pcr analysis of the galk colonies electroporated with the flag-gfp pcr product revealed that only 3/ <dig> colonies retained the galk cassette whereas 40/ <dig> colonies correctly have the flag-gfp tag instead.
to facilitate the use of modified fosmids for the creation of transgenic animals we exploited the loxp site in the fosmid backbone as a means to quickly insert selectable markers.
the sw <dig> bacterial strain used for recombineering also carries a transgene that expresses the cre recombinase under the control of the arabinose inducible arabad promoter  <cit> .
we constructed the plasmid ploxp unc- <dig> which carries the genomic dna that rescues the unc- <dig> mutant and a loxp site on the pmod <dig> vector backbone .
this plasmid is unable to replicate in sw <dig> due to the r6k replication origin.
we were able to integrate this plasmid into fosmids in sw <dig> by using electroporation followed by recovery in lb containing arabinose .
fosmids with successful integration were identified by selection on lb plates containing chloramphenicol and ampicillin.
the latter antibiotic selects for integration of the ampicillin resistance gene carried on the pmod <dig> backbone into the fosmid loxp site.
while the cre-lox recombination event is reversible, we have not found this to be a significant problem due to continued selection for ampicillin resistance and the use of the epi <dig> bacteria strain, which lacks cre recombinase, for large-scale growth and long-term storage of modified fosmids.
we tested whether the k10c <dig> ::gfp transgene created was functional by creating transgenic animals via biolistic bombardment.
we obtained a mix of transgenic animals carrying extrachromosomal arrays and integrated transgenes.
analysis of > <dig> lines revealed a similar pattern of gfp expression in the intestine and hypodermis .
additionally, the transgene produced a k10c <dig> :gfp fusion protein in vivo based on two lines of evidence.
first, rnai directed against either gfp or k10c <dig>  resulted in a loss of gfp expression as seen by fluorescence microscopy .
second, western blotting using α-flag antibodies, which detect a flag epitope at the start of gfp, identified a  <dig> kd protein consistent with a k10c <dig> :gfp fusion protein, and rnai directed at gfp or k10c <dig>  result in the loss of this protein .
together these data indicate the ability of recombineering to produce transgenes capable of producing fusion proteins and to introduce new epitope tags into target genes.
our work with k10c <dig>  suggested that the galk cassette was more robust for the negative selection step than the rt cassette.
to explore this observation, we created flag-gfp fusion genes for the dod- <dig> and myo- <dig> genes.
we found that similarly to k10c <dig> , the galk cassette proved more reliable in head-to-head testing with the rt cassette with regards the isolation of correctly modified fosmids after negative selection .
with the screening of  <dig> additional colonies, we found 1/ <dig> dod- <dig> and 2/ <dig> myo- <dig> colonies carrying flag-gfp for colonies created with the rt cassette whereas for the galk cassette 46/ <dig> dod- <dig> and 46/ <dig> myo- <dig> colonies carried flag-gfp .
remarkably, we found that colonies carrying flag-gfp could also be obtained with induced competent cells stored at -80°c for a full year .
besides the production of gfp fusion proteins, an increasing number of papers in c. elegans research are describing the use of transgenic worm strains for proteomic studies aimed at identifying protein interaction partners  <cit> .
one approach has been to use the tap tag which consists of a combination of protein-a and calmodulin binding peptide epitopes which allow the purification of tagged proteins at high purity via the use of two successive purification steps  <cit> .
the tap tag has been successfully used in c. elegans to purify the unc- <dig> acetylcholine receptor subunit along with multiple interacting proteins that were subsequently identified by mass spectrometry  <cit> .
to facilitate the creation of tap-tagged transgenes we modified pmod <dig> galk-g to add regions complementary to either the n-terminal or c-terminal version of the tap tag.
the resulting plasmid, pmod <dig> galk-gt, allows a single set of oligos to be used for the generation of the tagged fosmid through sequential rounds of recombineering .
discussion
a streamlined procedure for transgene production
we describe a modified protocol and accompanying vectors that can be used to easily produce fosmid-based transgenes for the generation of transgenic c. elegans.
our protocol involves three steps which are all carried out in the sw <dig> bacterial stain.
in the first step, the galk gene is integrated into the desired site in the fosmid to be modified through recombineering with a pcr product carrying galk flanked by homology arms complementary to the target fosmid .
we have improved this step relative to prior techniques via the integration of homology regions for either flag-gfp or a tap tag into the galk cassette.
this allows a single set of oligos to be used for the first and second steps of fosmid modification and lowers the cost of fosmid modification by 50%.
the integration of gfp homology arms into the rt cassette has also been recently described by bamps and hope  <cit> .
this modification is especially important if the technique is to be scaled up to genome-wide coverage.
we further simplify the first step through the use of a galk cassette carried on an r6k-based vector which is unable to replicate in the sw <dig> bacterial strain.
this improvement eliminates the need to either dpni digest nor extensively purify the pcr product used for recombineering.
in the second step, the galk cassette is replaced with tags such as gfp or tap which are then fused with the gene of interest on the fosmid .
the success of this step hinges on the effectiveness of the negative selection strategy used to identify correctly modified fosmids from those that are unmodified.
we found that the galk cassette was more reliable than the previously described rt cassette in head to head testing.
similar results have also been found by another lab .
this may be due to the galk gene product serving as the target of both positive and negative selection instead the two separate genes in the rt cassette which are involved in each step  <cit> .
the larger size of the rt cassette also required the use a lower fidelity polymerase, i.e.
a taq/proof-reading polymerase versus a proof-reading polymerase alone, for efficient amplification which may serve as a source of mutations in the cassette that could interfere with the negative selection step.
alternately, we have found that the rt cassette is somewhat toxic to bacteria which may lead to the selection for mutations that decrease the effectiveness of negative selection.
in contrast, for the galk cassette the positive selection step and subsequent re-streaking on macconkey agar select for robust galk expression before galk is used in the negative selection step.
additionally, our addition of the flag-gfp or tap homology regions into the galk cassette may also increase the efficiency of recombineering by increasing the length of the homology arms the pcr product to a maximum of  <dig> bp on each end.
however, we have failed to see a qualitative difference between the  <dig> bp homology arm and a shorter flag-gfp only pcr product with  <dig> bp homology arms when tested .
it still may be possible that for a subset of genes the  <dig> bp homology arms might make an important contribution.
finally, it may be possible that subtle changes in the protocol used by other groups may have a significant impact on the success of recombineering with the rt cassette as some groups have reported higher efficiency rates with it  <cit> .
in the third step a selectable marker for the generation of transgenic worms is added to the fosmid .
this creates a marker in cis and directly linked to the modified fosmid.
for the selectable marker, we chose the widely used unc- <dig> marker that is compatible with both microinjection and biolistic bombardment  <cit> .
bombardment is particularly useful as significant numbers of transgenic lines can be generated by a single trial and even inexperienced personnel can create transgenics .
as a result of our modifications, the entire procedure can be accomplished from start to production of a transgenic animal in a few weeks even by members of the lab who have a grasp of lab basic techniques but are not expert in molecular biology or microbiology.
applications
our lab has used this procedure to generate both gfp transgenes to examine gene expression pattern and response to rnai  <cit> .
we have also used this procedure to generate tap transgenes for proteomic studies, but these transgenic animals could also be used for chromatin or rna immunoprecipitation assays as well .
it might be possible to adapt this technique to liquid culture as means to create transgenes in a high throughput manner.
beyond the intended uses of this technique, we have found that recombineering provides the flexibility to generate alternate vectors at a later time.
specifically, the induced, competent bacteria carrying the galk intermediate can be stored for later use even months to a year later .
oligos carrying homology arms to the target gene can then be used to quickly add other tags generated by pcr.
for example, we have been able to generate a transgene carrying a modified tap tag which has been reported to produce higher purified protein yields in drosophila to conduct head-to-head tests  <cit> .
making this change only requires a few days of work to reach the stage of generating new transgenic animals.
finally while our work focused on applications in c. elegans, it is likely that some of these reagents can be used in the construction of modified bacs or fosmids for other experimental systems.
problems encountered
during construction of modified fosmids, we encountered problems with the modification of srp- <dig> and dod- <dig>  these fosmids are similar in that both contain multiple genes that are highly similar at the sequence level.
for example, the wrm0627ah <dig> fosmid contains both srp- <dig> and srp- <dig> and the wrm066cb <dig> fosmid contains dod- <dig> and k10d <dig> .
for srp- <dig> we discovered a rearrangement during the initial transformation into sw <dig>  this was easily detected via pcr using primers that anneal 50– <dig> nucleotides away on the genomic sequence flanking the desired modification site.
for dod- <dig> we found that the galk cassette integrated into another site on the fosmid.
this was similarly detected by pcr using flanking oligos.
both of these difficulties could be overcome by diligence during the screening of isolated colonies and are likely due to the presence of highly similar sequences that could lead to spurious recombination events.
to guard against moving forward with an incorrect fosmid, we have routinely screened colonies with both cassette-specific and flank sequence-specific oligos to ensure the right insert and right site.
the flanking oligos are also useful for the sequencing of the resulting fosmids.
CONCLUSIONS
we have modified existing recombineering protocols to create a simpler and more robust method of generating transgenes for use in the construction of transgenic worms.
the approach and vectors will be of benefit especially to smaller academic labs looking to begin to use fosmid-based transgenes.

 the generation of transgenic animals in model systems such as caenorhabditis elegans has created dramatic changes in the ability of researchers to approach biologic questions. it is relatively straightforward to create and use a transgenic animal to investigate the timing and pattern of gene expression, express epitope-tagged versions of genes, or test the effects of gene mis-expression. traditionally, constructs used for creation of a gfp fusion gene or epitope-tagged version of a gene were drawn from regions of cloned genomic dna which were known to rescue mutants of the specific gene. as the availability of rnai, microarrays, and other approaches which are based upon the worm genomic sequence instead of classical mutants has increased, a need to create transgenes in other ways has developed. for these genes, a region 5' of the gene is often isolated via pcr and then used as a promoter. this approach has been further simplified by the construction of a large number of vectors by the fire lab and later by the generation of the c. elegans promoterome. however, the decision about the exact length and location of the promoter is more often driven by arbitrary decisions about the size or convenient restriction sites than sequence analysis. as a result, it is difficult to know if the gfp expression pattern created by a selected promoter reflects the true expression pattern of the gene in vivo. to generate transgenes that are more likely to reflect the in vivo expression timing and pattern, interest in generating transgenics using the larger regions of genomic dna carried by either fosmids or bacs is growing. this approach carries the advantage of using 30– kb of genomic dna in the construction of a transgene so it is likely that all or most of the promoter and enhancer elements are included. additionally since the genomic dna for the gene is included, additional complexities such as alternative splicing, multiple transcription start sites, and regulation by 3' utr sequences or microrna can also occur. for this approach to become a standard technique within the c. elegans community, approaches need to be developed to make the modification of fosmids and bacs quick and robust for the novice. two recent publications have begun to lay the groundwork for this to occur. the first described the modification of c. elegans fosmids using homologous recombination in e. coli using pcr generated dna fragments. the creation of a transgene occurred through a two-step procedure where first a teta-rpsl cassette flanked by homology arms is inserted at the desired location and then in a second step the cassette is replaced by the desired dna, such as gfp, flanked by the same homology arms. this technique allows the insertion of essentially any sequence at any site in the gene. the second paper described the modification of bacs from the related nematode species caenorhabditis briggsae also by the use of recombineering. in this protocol, a gfp fusion gene is created by inserting gfp at the c-terminus of a gene of interest along with a flanking kanamycin resistance cassette via a single round of recombineering with a pcr product flanked by homology arms. the bac is also prepared for use as a transgene by insertion of the unc- gene via recombineering. the addition of the unc- gene provides an easily visualized selectable marker as this dna is able to rescue the size, body shape, mobility, and starvation resistance defects present in unc- mutants. each approach has strengths and weaknesses for the routine generation of transgenic worms. we have furthered their work by developing a streamlined series of procedures and accompanying vectors to allow the simple and reliable generation of transgenes ready for the transformation of worms by bombardment or microinjection. specifically, we focused on the use of c. elegans genomic dna cloned into a commercially available fosmid library. we then developed tools that will allow the robust and efficient generation of either flag-gfp or tap fusion genes by four important changes. first, we utilized the more robust galk gene for positive and then negative selection during recombineering. second, we constructed plasmids for galk utilizing the r6k origin which is unable to replicate in the e. coli strain used for recombineering and minimizes the amount of processing of the pcr products used. third, we built homology arms for both gfp and n and c-terminal tap tags into the galk cassette so that a single pair of oligos can be used for the generation of pcr products for each step. the oligos used for recombineering are long and require purification prior to use so the need for one pair instead of two reduces either the labor or cost involved in procuring the oligos. finally, we developed a protocol for adding the unc- gene to the fosmid backbone through cre-lox mediated recombination. the final fosmid product can then be used directly for the generation of transgenic nematodes.  we sought to use the c. elegans fosmid library and recombineering for the routine generation of transgenes and subsequent creation of transgenic worms. two techniques have been described using recombineering in c. elegans with one using a bac library generated from the related nematode species c. briggsae while the other used the fosmid library followed by microinjection of the modified plasmids. we initially used the protocol described by dolphin et. al. to successfully generate several daf- transgenes. during this process we identified several ways to optimize and simplify the protocol for recombineering and transgenic animal creation. the plasmid template used for the generation of the selection/counter-selection cassette by pcr, pbac-rt, is able to replicate in the sw bacterial strain used for recombineering. consequently, this template needs to be destroyed prior to electroporation either by dpni digestion or as suggested by dolphin et. al. through the use of a restriction fragment as the pcr template. as pbac-rt is constructed on a bac vector backbone, it is a very low copy number plasmid, and we experienced difficulty in purifying sufficient dna to digest and gel purify the fragment containing the rt cassette. to address this difficulty, we subcloned the rt cassette from pbac-rt into the pmod vector. pmod uses the r6k replication origin which requires the π protein for replication in e. coli. the resulting plasmid pmod rt has the advantages of having a higher copy number than pbac-rt which simplifies dna purification and of being unable to replicate in the sw bacterial strain. even when > ng. of pmod rt dna was electroporated into sw no tetr colonies were obtained. as a result, pcr products used for recombineering do not need to be digested with dpni or be extensively purified prior to use. for c. elegans experiments most transgenes consist of gfp fusions constructed to either identify the location and timing of gene expression and/or to visualize the cellular localization of the protein. to construct a gfp fusion transgene using the method of dolphin et. al. two pairs of long oligonucleotides need to be ordered and purified for the generation of a transgene. the first set anneal to the rt cassette and carry base pair homology arms specific for the gene of interest. the second set anneals to gfp and carry the same homology arms. if these are purchased from commercial companies the cost of these oligos is not insignificant especially if multiple transgenes are to be generated. to facilitate the construction of gfp fusion genes, we inserted oligos with base pair homology arms for gfp into pmod rt to generate pmod rt-g. the presence of these homology arms allows a single set of oligos to be used for the amplification of the rt cassette and gfp. this reduces the cost for oligos by half compared with the prior technique. during the construction of pmod rt and daf- transgenes using the rt cassette we found two limitations of the rt cassette. first, the rt cassette appears to be somewhat toxic to e. coli as pmod rt could only be maintained in the jm109λpir strain where it is a low copy number plasmid instead of the ec100pir- strain which increases the copy number of r6k based plasmids to ~ this toxicity may contribute to some of the difficulties we experienced with positive and negative selection of fosmids carrying the cassette. second, the rt cassette involves separate genes for positive and negative selection during recombineering. this property may increase the background during negative selection as mutations in the gene involved would be silent until the bacteria are plated on selective media. these mutations are most likely introduced during pcr amplification as amplification of the template would be expected to produce ~2–6% of products with mutations for proofreading polymerases and up to 25% for taq-proofreading polymerase blends. hence, any toxicity of the cassette would facilitate selection of mutated, less toxic clones. to address these two concerns we experimented with the use of the galk gene for positive and negative selection during recombineering. to facilitate the use of galk, we transferred the galk gene to the pmod plasmid and added gfp homology arms for use during recombineering. we initially compared the use of pmod rt-g and pmod galk-g by constructing a gfp fusion at the n-term of the k10c gene which encodes a member of the fumarylacetoacetate hydrolase family. prior attempts to create a gfp reporter using the promoterome library clone were unsuccessful perhaps due to a lack of important promoter elements in the promoter clone. we found that the rt and galk cassettes can be easily inserted into a fosmid carrying k10c by recombineering. we then used a pcr fragment carrying flag-tagged gfp to replace either the rt or galk cassette via a second round of recombineering. colonies with successful substitution of flag-gfp were identified by using selective media. we found the galk cassette was more robust than the rt cassette as 4/ colonies created with the galk cassette and 0/ colonies created with the rt cassette carried flag-gfp by pcr. pcr analysis of a further clones revealed 40/ colonies created with the galk cassette and 0/ colonies created with the rt cassette carried flag-gfp after selection. dod-22 myo-3 the source of this difference appears to be at least in part due to weaker negative selection from the rt cassette as revealed by analysis of colonies obtained after a no pcr product control electroporation. this control was conducted similarly to the second recombineering step but no flag-gfp pcr product was added prior to electroporation. pcr analysis of colonies for the galk and rt cassettes revealed that 23/ galk cassette bacteria still had at least part of the cassette whereas 46/ rt colonies still had a cassette detectable by pcr. this suggests that a significant number of bacteria with the rt cassette are still able to grow despite negative selection. further, addition of the flag-gfp pcr product to the electroporation resulted in a noticeable increase in colonies from bacteria with the galk cassette but not from bacteria with the rt cassette. these counts were performed in parallel and included bacteria from undiluted, 1: diluted, and 1: diluted samples plated from a single experiment. together, the greater number of total colonies obtained from bacteria with the galk cassette along with the more effective negative selection might lead to the greater effectiveness of the galk cassette in recombineering as the significant number of true positives could now outnumber the relatively small number of false positives. consistent with this, pcr analysis of the galk colonies electroporated with the flag-gfp pcr product revealed that only 3/ colonies retained the galk cassette whereas 40/ colonies correctly have the flag-gfp tag instead. to facilitate the use of modified fosmids for the creation of transgenic animals we exploited the loxp site in the fosmid backbone as a means to quickly insert selectable markers. the sw bacterial strain used for recombineering also carries a transgene that expresses the cre recombinase under the control of the arabinose inducible arabad promoter. we constructed the plasmid ploxp unc- which carries the genomic dna that rescues the unc- mutant and a loxp site on the pmod vector backbone. this plasmid is unable to replicate in sw due to the r6k replication origin. we were able to integrate this plasmid into fosmids in sw by using electroporation followed by recovery in lb containing arabinose. fosmids with successful integration were identified by selection on lb plates containing chloramphenicol and ampicillin. the latter antibiotic selects for integration of the ampicillin resistance gene carried on the pmod backbone into the fosmid loxp site. while the cre-lox recombination event is reversible, we have not found this to be a significant problem due to continued selection for ampicillin resistance and the use of the epi bacteria strain, which lacks cre recombinase, for large-scale growth and long-term storage of modified fosmids. we tested whether the k10c::gfp transgene created was functional by creating transgenic animals via biolistic bombardment. we obtained a mix of transgenic animals carrying extrachromosomal arrays and integrated transgenes. analysis of > lines revealed a similar pattern of gfp expression in the intestine and hypodermis. additionally, the transgene produced a k10c:gfp fusion protein in vivo based on two lines of evidence. first, rnai directed against either gfp or k10c resulted in a loss of gfp expression as seen by fluorescence microscopy. second, western blotting using α-flag antibodies, which detect a flag epitope at the start of gfp, identified a kd protein consistent with a k10c:gfp fusion protein, and rnai directed at gfp or k10c result in the loss of this protein. together these data indicate the ability of recombineering to produce transgenes capable of producing fusion proteins and to introduce new epitope tags into target genes. our work with k10c suggested that the galk cassette was more robust for the negative selection step than the rt cassette. to explore this observation, we created flag-gfp fusion genes for the dod- and myo- genes. we found that similarly to k10c, the galk cassette proved more reliable in head-to-head testing with the rt cassette with regards the isolation of correctly modified fosmids after negative selection. with the screening of additional colonies, we found 1/ dod- and 2/ myo- colonies carrying flag-gfp for colonies created with the rt cassette whereas for the galk cassette 46/ dod- and 46/ myo- colonies carried flag-gfp. remarkably, we found that colonies carrying flag-gfp could also be obtained with induced competent cells stored at -80°c for a full year. besides the production of gfp fusion proteins, an increasing number of papers in c. elegans research are describing the use of transgenic worm strains for proteomic studies aimed at identifying protein interaction partners. one approach has been to use the tap tag which consists of a combination of protein-a and calmodulin binding peptide epitopes which allow the purification of tagged proteins at high purity via the use of two successive purification steps. the tap tag has been successfully used in c. elegans to purify the unc- acetylcholine receptor subunit along with multiple interacting proteins that were subsequently identified by mass spectrometry. to facilitate the creation of tap-tagged transgenes we modified pmod galk-g to add regions complementary to either the n-terminal or c-terminal version of the tap tag. the resulting plasmid, pmod galk-gt, allows a single set of oligos to be used for the generation of the tagged fosmid through sequential rounds of recombineering. discussion a streamlined procedure for transgene production we describe a modified protocol and accompanying vectors that can be used to easily produce fosmid-based transgenes for the generation of transgenic c. elegans. our protocol involves three steps which are all carried out in the sw bacterial stain. in the first step, the galk gene is integrated into the desired site in the fosmid to be modified through recombineering with a pcr product carrying galk flanked by homology arms complementary to the target fosmid. we have improved this step relative to prior techniques via the integration of homology regions for either flag-gfp or a tap tag into the galk cassette. this allows a single set of oligos to be used for the first and second steps of fosmid modification and lowers the cost of fosmid modification by 50%. the integration of gfp homology arms into the rt cassette has also been recently described by bamps and hope. this modification is especially important if the technique is to be scaled up to genome-wide coverage. we further simplify the first step through the use of a galk cassette carried on an r6k-based vector which is unable to replicate in the sw bacterial strain. this improvement eliminates the need to either dpni digest nor extensively purify the pcr product used for recombineering. in the second step, the galk cassette is replaced with tags such as gfp or tap which are then fused with the gene of interest on the fosmid. the success of this step hinges on the effectiveness of the negative selection strategy used to identify correctly modified fosmids from those that are unmodified. we found that the galk cassette was more reliable than the previously described rt cassette in head to head testing. similar results have also been found by another lab. this may be due to the galk gene product serving as the target of both positive and negative selection instead the two separate genes in the rt cassette which are involved in each step. the larger size of the rt cassette also required the use a lower fidelity polymerase, i.e. a taq/proof-reading polymerase versus a proof-reading polymerase alone, for efficient amplification which may serve as a source of mutations in the cassette that could interfere with the negative selection step. alternately, we have found that the rt cassette is somewhat toxic to bacteria which may lead to the selection for mutations that decrease the effectiveness of negative selection. in contrast, for the galk cassette the positive selection step and subsequent re-streaking on macconkey agar select for robust galk expression before galk is used in the negative selection step. additionally, our addition of the flag-gfp or tap homology regions into the galk cassette may also increase the efficiency of recombineering by increasing the length of the homology arms the pcr product to a maximum of bp on each end. however, we have failed to see a qualitative difference between the bp homology arm and a shorter flag-gfp only pcr product with bp homology arms when tested. it still may be possible that for a subset of genes the bp homology arms might make an important contribution. finally, it may be possible that subtle changes in the protocol used by other groups may have a significant impact on the success of recombineering with the rt cassette as some groups have reported higher efficiency rates with it. in the third step a selectable marker for the generation of transgenic worms is added to the fosmid. this creates a marker in cis and directly linked to the modified fosmid. for the selectable marker, we chose the widely used unc- marker that is compatible with both microinjection and biolistic bombardment. bombardment is particularly useful as significant numbers of transgenic lines can be generated by a single trial and even inexperienced personnel can create transgenics. as a result of our modifications, the entire procedure can be accomplished from start to production of a transgenic animal in a few weeks even by members of the lab who have a grasp of lab basic techniques but are not expert in molecular biology or microbiology. applications our lab has used this procedure to generate both gfp transgenes to examine gene expression pattern and response to rnai. we have also used this procedure to generate tap transgenes for proteomic studies, but these transgenic animals could also be used for chromatin or rna immunoprecipitation assays as well. it might be possible to adapt this technique to liquid culture as means to create transgenes in a high throughput manner. beyond the intended uses of this technique, we have found that recombineering provides the flexibility to generate alternate vectors at a later time. specifically, the induced, competent bacteria carrying the galk intermediate can be stored for later use even months to a year later. oligos carrying homology arms to the target gene can then be used to quickly add other tags generated by pcr. for example, we have been able to generate a transgene carrying a modified tap tag which has been reported to produce higher purified protein yields in drosophila to conduct head-to-head tests. making this change only requires a few days of work to reach the stage of generating new transgenic animals. finally while our work focused on applications in c. elegans, it is likely that some of these reagents can be used in the construction of modified bacs or fosmids for other experimental systems. problems encountered during construction of modified fosmids, we encountered problems with the modification of srp- and dod- these fosmids are similar in that both contain multiple genes that are highly similar at the sequence level. for example, the wrm0627ah fosmid contains both srp- and srp- and the wrm066cb fosmid contains dod- and k10d. for srp- we discovered a rearrangement during the initial transformation into sw this was easily detected via pcr using primers that anneal 50– nucleotides away on the genomic sequence flanking the desired modification site. for dod- we found that the galk cassette integrated into another site on the fosmid. this was similarly detected by pcr using flanking oligos. both of these difficulties could be overcome by diligence during the screening of isolated colonies and are likely due to the presence of highly similar sequences that could lead to spurious recombination events. to guard against moving forward with an incorrect fosmid, we have routinely screened colonies with both cassette-specific and flank sequence-specific oligos to ensure the right insert and right site. the flanking oligos are also useful for the sequencing of the resulting fosmids.  we have modified existing recombineering protocols to create a simpler and more robust method of generating transgenes for use in the construction of transgenic worms. the approach and vectors will be of benefit especially to smaller academic labs looking to begin to use fosmid-based transgenes. 
among these are the use of galk gene as a selection marker for both the positive and negative selection steps in recombineering, the use of r6k based plasmids which eliminate the need for extensive pcr product purification, a means to integrate the unc- marker on to the fosmid backbone, and placement of homology arms to commonly used gfp and tap fusion genes flanking the galk cassette which reduces the cost of oligos by 50%. the generation of transgenic animals is an important experimental tool and allows use of gfp fusion proteins to study the expression of genes of interest or generation of epitope tagged versions of specific genes. to better capture the genuine expression pattern and timing, several investigators have modified large pieces of dna carried by bacs or fosmids for use in the construction of transgenic animals via recombineering. we have made several significant changes that allow the production of c. elegans transgenes from a commercially available fosmid library in a robust and streamlined manner.
 the nematode caenorhabditis elegans has emerged as a powerful system to study biologic questions ranging from development to aging. the generation of transgenic animals is an important experimental tool and allows use of gfp fusion proteins to study the expression of genes of interest or generation of epitope tagged versions of specific genes. transgenes are often generated by placing a promoter upstream of a reporter gene or cdna. this often produces a representative expression pattern, but important exceptions have been observed. to better capture the genuine expression pattern and timing, several investigators have modified large pieces of dna carried by bacs or fosmids for use in the construction of transgenic animals via recombineering. however, these techniques are not in widespread use despite the advantages when compared to traditional approaches. additionally, some groups have encountered problems with employing these techniques. hence, we sought identify ways to improve the simplicity and reliability of the procedure.  we describe here several important modifications we have made to existing protocols to make the procedure simpler and more robust. among these are the use of galk gene as a selection marker for both the positive and negative selection steps in recombineering, the use of r6k based plasmids which eliminate the need for extensive pcr product purification, a means to integrate the unc- marker on to the fosmid backbone, and placement of homology arms to commonly used gfp and tap fusion genes flanking the galk cassette which reduces the cost of oligos by 50%.  we have made several significant changes that allow the production of c. elegans transgenes from a commercially available fosmid library in a robust and streamlined manner. these changes make the technique more attractive especially to small academic labs unfamiliar with recombineering. 