期刊
GENOMICS
卷 95, 期 1, 页码 37-46出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ygeno.2009.09.001
关键词
Biolistic transformation; Gene knockout; Homologous recombination; Flp recombinase; Phospholipase C
资金
- NIH National Center for Research Resources
- BBSRC
- Biotechnology and Biological Sciences Research Council [BB/C507661/1] Funding Source: researchfish
- Medical Research Council [G117/466] Funding Source: researchfish
- MRC [G117/466] Funding Source: UKRI
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 market, 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. (C) 2009 Elsevier Inc. All rights reserved.
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