RRC ID 3964
Author Zhang Y, Nash L, Fisher AL.
Title A simplified, robust, and streamlined procedure for the production of C. elegans transgenes via recombineering.
Journal BMC Dev. Biol.
Abstract BACKGROUND: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.
RESULTS: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-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%.
CONCLUSION: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.
Volume 8
Pages 119
Published 2008-12-30
DOI 10.1186/1471-213X-8-119
PII 1471-213X-8-119
PMID 19116030
PMC PMC2629773
MeSH Animals Animals, Genetically Modified / genetics* Caenorhabditis elegans / genetics* Caenorhabditis elegans / metabolism Caenorhabditis elegans Proteins / genetics Caenorhabditis elegans Proteins / metabolism Genetic Engineering / methods* Plasmids / genetics Plasmids / metabolism RNA Interference Recombinant Fusion Proteins / genetics Recombinant Fusion Proteins / metabolism Transgenes*
IF 2.427
Times Cited 21
WOS Category DEVELOPMENTAL BIOLOGY
Resource
Prokaryotes E. coli CV-43(JM109(Lambdapir))