Generating transgenic nematodes by bombardment and antibiotic selection

Semple, Jennifer I.; Biondini, Laura; Lehner, Ben

doi:10.1038/nmeth.1864

Cited by 34 publications

(31 citation statements)

References 6 publications

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“…As no commercial antibody against PTL-1 is available, we tagged PTL-1 at the C-terminus with the short peptide V5 in order to enable detection of PTL-1 expressed from the transgene. Transgenic worms were generated by biolistic transformation, and an integrated line was obtained after selection using the dual antibiotic method (Semple et al, 2012). We confirmed that our transgene was expressed by immunoblotting ( Fig.…”

Section: Resultssupporting

confidence: 54%

“…Wild-type worms were bombarded with 7 mg of linearised plasmid DNA using previously established methods (Praitis et al, 2001). Selection post-bombardment was undertaken using the dual antibiotic selection protocol (Semple et al, 2012). Integrated lines expressing htau40 cDNA and PTL-1 cDNA tagged at the carboxyl (C)-terminus with V5 under the control of the ptl-1 promoter and PTL-1 39UTR were obtained and outcrossed six times to wild-type.…”

Section: Generation Of Transgenic Linesmentioning

confidence: 99%

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Protein with tau-like repeats regulates neuronal integrity and lifespan inC. elegans

Chew¹,

Fan²,

Götz³

et al. 2013

Journal of Cell Science

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SummaryProtein with tau-like repeats (PTL-1) is the sole Caenorhabditis elegans homolog of tau and MAP2, which are members of the mammalian family of microtubule-associated proteins (MAPs). In mammalian neurons, tau and MAP2 are segregated, with tau being mainly localised to the axon and MAP2 mainly to the dendrite. In particular, tau plays a crucial role in pathology, as elevated levels lead to the formation of tau aggregates in many neurodegenerative conditions including Alzheimer's disease. We used PTL-1 in C. elegans to model the biological functions of a tau-like protein without the complication of functional redundancy that is observed among the mammalian MAPs. Our findings indicate that PTL-1 is important for the maintenance of neuronal health as animals age, as well as in the regulation of whole organism lifespan. In addition, gene dosage of PTL-1 is crucial because variations from wild-type levels are detrimental. We also observed that human tau is unable to robustly compensate for loss of PTL-1, although phenotypes observed in tau transgenic worms are dependent on the presence of endogenous PTL-1. Our data suggest that some of the effects of tau pathology result from the loss of physiological tau function and not solely from a toxic gain-of-function due to accumulation of tau.

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Section: Resultssupporting

confidence: 54%

Section: Generation Of Transgenic Linesmentioning

confidence: 99%

Protein with tau-like repeats regulates neuronal integrity and lifespan inC. elegans

Chew¹,

Fan²,

Götz³

et al. 2013

Journal of Cell Science

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“…The full length ctbp-1a cDNA sequence (2184 bp) amplified from the pGC188-ctbp-1a::V5 plasmid and ctbp-1 3′ UTR sequence (574 bp) amplified from the fosmid WRM0622cG05 (Source Bioscience) which contains the ctbp-1 gene were incorporated into the Gateway® pDONR™221 and pDONR™P2R-P3 vectors (Life Technologies, Australia) respectively using the Gateway® Cloning system. A LR reaction was performed with the following plasmids: pDONR p dpy-7, pDONR ctbp-1a::V5, pDONR ctbp-1 3′ UTR and the destination plasmid, pBCN40-R4R3 (Addgene plasmid 34915) (Semple et al, 2012). The resulting plasmid pAER017 p dpy-7::ctbp-1a::V5::ctbp-1 3′ UTR was linearised by digestion with BamHI and~10 μg used for biolistic transformation into wild type animals.…”

Section: Generating a Transgenic Strain For Expression Of Ctbp-1a In mentioning

confidence: 99%

The transcriptional repressor CTBP-1 functions in the nervous system of Caenorhabditis elegans to regulate lifespan

Reid

Yücel

Wood

et al. 2014

Experimental Gerontology

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“…Using axenic mCeHR liquid culture allows more efficient growth and subsequently more worms for bombardments. Additionally, bombardments can be used in conjunction with drug selection to avoid using unc-119 (ed3) worms 21 .…”

Section: Discussionmentioning

confidence: 99%

Culturing <em>Caenorhabditis elegans</em > in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

Samuel

Sinclair

Pinter

et al. 2014

JoVE

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In this protocol, we present the required materials, and the procedure for making modified C. elegans Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment. Video LinkThe video component of this article can be found at

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Generating transgenic nematodes by bombardment and antibiotic selection

Cited by 34 publications

References 6 publications

Protein with tau-like repeats regulates neuronal integrity and lifespan inC. elegans

Protein with tau-like repeats regulates neuronal integrity and lifespan inC. elegans

The transcriptional repressor CTBP-1 functions in the nervous system of Caenorhabditis elegans to regulate lifespan

Culturing <em>Caenorhabditis elegans</em > in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

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