The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker
Summary
The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.
Abstract
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.
Protocol
Discussion
The generation of transgenes from fosmids offers the benefit of retaining all of the native promoter elements, splice variants, and 3′ UTR regulatory elements. This can lead to the construction of a transgene which is more reflective of the native expression pattern, or the construction of a functional transgene when other approaches fail 5. The resulting transgenes can carry a variety of epitope tags including GFP or a TAP tag.
The construction of transgenes involved three steps w…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors would like to thank Lindsey Nash for help with developing the technique. This work was funded by NIH grant AG028977 to A.L.F., a pilot project grant from the University of Pittsburgh OAIC (AG024827), and seed funds from the University of Pittsburgh.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| FosmidMAX kit | Epicentre | FMAX046 | ||
| GoTaq | Promega | M7122 | ||
| MOPS Media | Teknova | M2120 | ||
| 0.132 M Potassium phosphate solution | Teknova | M2102 | ||
| D-galactose | Sigma | G0750 | ||
| 2-deoxygalactose | Sigma | D4407 | ||
| Biotin | Sigma | B4639 | ||
| Leucine | Sigma | L8000 | ||
| NH4Cl | Sigma | A9434 | ||
| Phusion DNA polymerase | NEB | F-530S | ||
| MacConkey agar base | Becton Dickinson | 281810 | ||
| Arabinose | Sigma | A3131 | ||
| Chloramphenicol | Sigma | C1919 | ||
| Sodium phosphate dibasic | Sigma | S5136 | ||
| Potassium phosphate monobasic | Sigma | P5655 | ||
| Sodium chloride | Sigma | S5886 | ||
| Glycerol | Sigma | G2025 | ||
| Bacto Agar | Becton Dickinson | 214010 |
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