Reference - Detail
| RRC ID | 46743 |
|---|---|
| Author | Vázquez-Manrique RP, Legg JC, Olofsson B, Ly S, Baylis HA. |
| Title | Improved gene targeting in C. elegans using counter-selection and Flp-mediated marker excision. |
| Journal | Genomics |
| Abstract |
Gene targeting is widely used for the precise manipulation of genes. However, in the model organism Caenorhabditis elegans non-transposon mediated gene targeting remains laborious, and as a result has not been widely used. One obstacle to the wider use of this approach is the difficulty of identifying homologous recombination events amongst non-specific events. To improve gene targeting in C. elegans, we used a counter-selection approach to reduce the number of false positives; this involved using unc-119 as a positive-selection marker and GFP as a counter-selection marker which is lost during homologous recombination. This method of gene targeting allows straightforward screening for homologous events using a dissecting microscope equipped for fluorescence. In addition, to improve the final engineered product, we utilised Flp recombinase to remove the unc-119 selection marker, in somatic cells, producing clean knockouts in these cells. Using this strategy we have produced a knockout of the plc-4 gene, which encodes phospholipase C-delta in C. elegans, and demonstrated that conditional gene knockout is feasible in C. elegans. |
| Volume | 95(1) |
| Pages | 37-46 |
| Published | 2010-1-1 |
| DOI | 10.1016/j.ygeno.2009.09.001 |
| PII | S0888-7543(09)00207-9 |
| PMID | 19747540 |
| MeSH | Animals Animals, Genetically Modified Caenorhabditis elegans / cytology Caenorhabditis elegans / genetics* Caenorhabditis elegans / metabolism Caenorhabditis elegans Proteins / genetics Caenorhabditis elegans Proteins / metabolism DNA Nucleotidyltransferases / metabolism* Gene Expression Gene Knockout Techniques / methods* Genes, Helminth Genetic Markers Green Fluorescent Proteins Microscopy, Confocal Microscopy, Fluorescence Nerve Tissue Proteins / genetics Nerve Tissue Proteins / metabolism Phospholipase C delta / deficiency Phospholipase C delta / genetics Recombination, Genetic |
| IF | 6.205 |
| Times Cited | 12 |
| WOS Category | BIOTECHNOLOGY & APPLIED MICROBIOLOGY GENETICS & HEREDITY |
| Altmetric score |
オルトメトリクス指標項目
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| Total number of mentions | 0 |
| Altmetric score changes over past 6months | 0.0 |
| Resource | |
| C.elegans | tm1521 |