A simplified, robust, and streamlined procedure for the production of C. eleganstransgenes via recombineering

Yue Zhang,Alfred L Fisher,Lindsey Nash

doi:10.1186/1471-213x-8-119

Abstract

BackgroundThe 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.ResultsWe 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-119 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%.ConclusionWe 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.

Highlights

The nematode Caenorhabditis elegans has emerged as a powerful system to study biologic questions ranging from development to aging
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 [1]
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 [6,7]

Summary

Introduction

The nematode Caenorhabditis elegans has emerged as a powerful system to study biologic questions ranging from development to aging. 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. These techniques are not in widespread use despite the advantages when compared to traditional approaches. 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 used as a promoter [2,3]. 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 [1]

Methods

Results

Discussion

Conclusion