The creation of transgenic animals is widely utilized in C. elegans research 
including the use of GFP fusion proteins to study the regulation and expression 
pattern of genes of interest or generation of tandem affinity purification (TAP) 
tagged versions of specific genes to facilitate their purification. Typically 
transgenes are generated by placing a promoter upstream of a GFP reporter gene 
or cDNA of interest, and this often produces a representative expression 
pattern. However, critical elements of gene regulation, such as control elements 
in the 3' untranslated region or alternative promoters, could be missed by this 
approach. Further only a single splice variant can be usually studied by this 
means. In contrast, the use of worm genomic DNA carried by fosmid DNA clones 
likely includes most if not all elements involved in gene regulation in vivo 
which permits the greater ability to capture the genuine expression pattern and 
timing. To facilitate the generation of transgenes using fosmid DNA, we describe 
an E. coli based recombineering procedure to insert GFP, a TAP-tag, or other 
sequences of interest into any location in the gene. The procedure uses the galK 
gene as the selection marker for both the positive and negative selection steps 
in recombineering which results in obtaining the desired modification with high 
efficiency. Further, plasmids containing the galK gene flanked by homology arms 
to commonly used GFP and TAP fusion genes are available which reduce the cost of 
oligos by 50% when generating a GFP or TAP fusion protein. These plasmids use 
the R6K replication origin which precludes the need for extensive PCR product 
purification. Finally, we also demonstrate a technique to integrate the unc-119 
marker on to the fosmid backbone which allows the fosmid to be directly injected 
or bombarded into worms to generate transgenic animals. This video demonstrates 
the procedures involved in generating a transgene via recombineering using this 
method.