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

Zhang Y, Nash L, Fisher AL - BMC Dev. Biol. (2008)

Bottom Line: However, these techniques are not in widespread use despite the advantages when compared to traditional approaches.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Pittsburgh, Department of Medicine, Division of Geriatric Medicine and Pittsburgh Institute for Neurodegenerative Diseases, Pittsburgh, PA 15260, USA. yzhang1@bidmc.harvard.edu

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.

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Related in: MedlinePlus

Generation of transgenic animals with modified fosmids. The modified fosmid carrying the K10C2.4:FLAG-GFP transgene was introduced into the DP38 (unc-119(ed3)) strain via microparticle bombardment. (A) GFP expression seen in the transgenic worms. (B and C) GFP expression is reduced by treatment with GFP (B) or K10C2.4 (C) RNAi. (D) Detection of the FLAG-GFP transgene in RNAi treated worm extracts by western blotting with anti-FLAG antibodies. Equal loading was confirmed by blotting with anti-actin antibodies.
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Figure 4: Generation of transgenic animals with modified fosmids. The modified fosmid carrying the K10C2.4:FLAG-GFP transgene was introduced into the DP38 (unc-119(ed3)) strain via microparticle bombardment. (A) GFP expression seen in the transgenic worms. (B and C) GFP expression is reduced by treatment with GFP (B) or K10C2.4 (C) RNAi. (D) Detection of the FLAG-GFP transgene in RNAi treated worm extracts by western blotting with anti-FLAG antibodies. Equal loading was confirmed by blotting with anti-actin antibodies.

Mentions: We tested whether the K10C2.4::GFP transgene created was functional by creating transgenic animals via biolistic bombardment. We obtained a mix of transgenic animals carrying extrachromosomal arrays and integrated transgenes. Analysis of >15 lines revealed a similar pattern of GFP expression in the intestine and hypodermis (Figure 4A and data not shown). Additionally, the transgene produced a K10C2.4:GFP fusion protein in vivo based on two lines of evidence. First, RNAi directed against either GFP or K10C2.4 resulted in a loss of GFP expression as seen by fluorescence microscopy (Figure 4B and 4C). Second, western blotting using α-FLAG antibodies, which detect a FLAG epitope at the start of GFP, identified a 76 kD protein consistent with a K10C2.4:GFP fusion protein, and RNAi directed at GFP or K10C2.4 result in the loss of this protein (Figure 4D). Together these data indicate the ability of recombineering to produce transgenes capable of producing fusion proteins and to introduce new epitope tags into target genes.


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

Zhang Y, Nash L, Fisher AL - BMC Dev. Biol. (2008)

Generation of transgenic animals with modified fosmids. The modified fosmid carrying the K10C2.4:FLAG-GFP transgene was introduced into the DP38 (unc-119(ed3)) strain via microparticle bombardment. (A) GFP expression seen in the transgenic worms. (B and C) GFP expression is reduced by treatment with GFP (B) or K10C2.4 (C) RNAi. (D) Detection of the FLAG-GFP transgene in RNAi treated worm extracts by western blotting with anti-FLAG antibodies. Equal loading was confirmed by blotting with anti-actin antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2629773&req=5

Figure 4: Generation of transgenic animals with modified fosmids. The modified fosmid carrying the K10C2.4:FLAG-GFP transgene was introduced into the DP38 (unc-119(ed3)) strain via microparticle bombardment. (A) GFP expression seen in the transgenic worms. (B and C) GFP expression is reduced by treatment with GFP (B) or K10C2.4 (C) RNAi. (D) Detection of the FLAG-GFP transgene in RNAi treated worm extracts by western blotting with anti-FLAG antibodies. Equal loading was confirmed by blotting with anti-actin antibodies.
Mentions: We tested whether the K10C2.4::GFP transgene created was functional by creating transgenic animals via biolistic bombardment. We obtained a mix of transgenic animals carrying extrachromosomal arrays and integrated transgenes. Analysis of >15 lines revealed a similar pattern of GFP expression in the intestine and hypodermis (Figure 4A and data not shown). Additionally, the transgene produced a K10C2.4:GFP fusion protein in vivo based on two lines of evidence. First, RNAi directed against either GFP or K10C2.4 resulted in a loss of GFP expression as seen by fluorescence microscopy (Figure 4B and 4C). Second, western blotting using α-FLAG antibodies, which detect a FLAG epitope at the start of GFP, identified a 76 kD protein consistent with a K10C2.4:GFP fusion protein, and RNAi directed at GFP or K10C2.4 result in the loss of this protein (Figure 4D). Together these data indicate the ability of recombineering to produce transgenes capable of producing fusion proteins and to introduce new epitope tags into target genes.

Bottom Line: However, these techniques are not in widespread use despite the advantages when compared to traditional approaches.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Pittsburgh, Department of Medicine, Division of Geriatric Medicine and Pittsburgh Institute for Neurodegenerative Diseases, Pittsburgh, PA 15260, USA. yzhang1@bidmc.harvard.edu

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.

Show MeSH
Related in: MedlinePlus