Selectable genetic markers for nematode transgenesis - PubMed (original) (raw)

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Selectable genetic markers for nematode transgenesis

Rosina Giordano-Santini et al. Cell Mol Life Sci. 2011 Jun.

Abstract

The nematode Caenorhabditis elegans has been used to study genetics and development since the mid-1970s. Over the years, the arsenal of techniques employed in this field has grown steadily in parallel with the number of researchers using this model. Since the introduction of C. elegans transgenesis, nearly 20 years ago, this system has been extensively used in areas such as rescue experiments, gene expression studies, and protein localization. The completion of the C. elegans genome sequence paved the way for genome-wide studies requiring higher throughput and improved scalability than provided by traditional genetic markers. The development of antibiotic selection systems for nematode transgenesis addresses these requirements and opens the possibility to apply transgenesis to investigate biological functions in other nematode species for which no genetic markers had been developed to date.

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Figures

Fig. 1

Fig. 1

Comparison between traditional non-selectable genetic markers (left) and antibiotic resistance markers (right) in the context of microinjection. When visual phenotypic markers are used for nematode transgenesis by microinjection, transgenic F1 worms need to be screened and isolated by hand by an experimentalist (left), while antibiotic selection offers the possibility of hands-off selection and maintenance of stable transgenic lines (right). In the case of antibiotic resistance markers, F1 isolation by hand ensures clonal homogeneity of isolated strains, but further maintenance can be done by chunking without further visual screening as long as the animals are kept on antibiotic-containing medium. *In the presence of antibiotic, wild-type larvae are unable to develop and die after a few days

Fig. 2

Fig. 2

Hands-off maintenance of an extrachromosomal C. elegans line on antibiotic-selective medium. An extrachromosomal C. elegans line carrying the NeoR nematode cassette and a p _myo_-2::gfp reporter (worms carrying p _myo_-2::gfp express GFP in the pharynx) amplified on a plate without G-418 (left) and on a plate supplemented with G-418 at the critical concentration (selective plate) (right). After several generations and in the absence of a selective agent, the extrachromosomal array is lost and transgenic animals are rare (left) while on selective plates wild-type worms are eliminated at the L1 stage and the transgenic population is enriched without manual maintenance

Fig. 3a–d

Fig. 3a–d

Transgene integration by Mos1 single-copy insertion (_Mos_SCI) mechanism. The main steps of _Mos_SCI are represented. On the right is a schematic view of the expression profile of the worm at each step. a Extrachromosomal array formation: a C. elegans strain carrying a single characterized insertion of Mos1 is injected with a transposase source, a repair template vector, and genetic markers for the identification of worms carrying the extrachromosomal array. The repair template is composed of the gene of interest and a co-insertion marker, both flanked by 1.5 kb of genomic DNA corresponding to the right (R) and left (L) sequences flanking the Mos1 insertion locus. b Expression of the transposase: Mos1 excision by the transposase creates a double-strand break in the genomic DNA. c Gene conversion: the double-strand break is repaired by gene conversion using the repair template in the extrachromosomal array, leading to the insertion of the gene of interest and the co-insertion marker into the genome. d Loss of the extrachromosomal array: insertion events are discriminated from nonintegrated transgenes by identifying animals expressing the co-insertion marker but lacking the transformation markers contained solely in the extrachromosomal array. Adapted from [41]

Fig. 4a–d

Fig. 4a–d

Comparison between _Mos_SCI using _unc_-119 as co-insertion marker (left) and _Mos_SCI-biotic (right). The use of antibiotic resistance genes as co-insertion markers facilitates almost every step of _Mos_SCI. a, b Every _Mos_1 recipient strain from NemaGENETAG can be used without the need to cross into an _unc_-119 mutant background, making host strains easier to amplify and to inject. c When using the transposase source under the control of a heat-shock promoter, large populations of nonintegrated adults can be easily obtained for heat-shock by hands-off enrichment on selective medium. d When using an antibiotic resistance gene as co-insertion marker, screening for insertion events is done in smaller populations that are not crowded by nontransgenic worms

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