The production of C. elegans transgenes via recombineering with the galK selectable marker.
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.
Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, USA. yzhang1@bidmc.harvard.edu
Yue
Y
Zhang
Luv
L
Kashyap
Annabel A
AA
Ferguson
Alfred L
AL
Fisher
D000818:Animals; D030801:Animals, Genetically Modified; D017173:Caenorhabditis elegans / Q000201:enzymology / Q000235:genetics*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics; D005686:Galactokinase / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D005819:Genetic Markers; D011993:Recombinant Fusion Proteins / Q000235:genetics; D011995:Recombination, Genetic; D019076:Transgenes
D029742:Caenorhabditis elegans Proteins; D005819:Genetic Markers; D011993:Recombinant Fusion Proteins; D005686:Galactokinase
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