Efficient generation of gene knockout plasmids for Dictyostelium discoideum using one-step cloning.
Genomics
; 97(5): 321-5, 2011 May.
Article
em En
| MEDLINE
| ID: mdl-21316445
ABSTRACT
The amoeba Dictyostelium discoideum is a well-established model organism for studying numerous aspects of cellular and developmental functions. Its rather small (~34Mb) chromosomal genome and the high efficiency of gene disruption by homologous recombination have enabled researchers to dissect various specific gene functions. We describe here the use of one-step cloning for the fast and efficient generation of deletion vectors that are produced in a one-step reaction by inserting two PCR products into an organism-specific, generic acceptor system. This worked efficiently for all 16 tested constructs directed against genes in the amoeba Dictyostelium discoideum. Saving cost and time, the used protocol represents a significant advancement in the generation of such plasmids compared to the conventionally applied restriction enzyme/ligation approach. Using appropriate selection markers, similar systems could also be useful in other organisms, where genes can be knocked out by homologous recombination.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Plasmídeos
/
Clonagem Molecular
/
Deleção de Genes
/
Dictyostelium
/
Técnicas de Inativação de Genes
Tipo de estudo:
Evaluation_studies
Limite:
Animals
Idioma:
En
Revista:
Genomics
Assunto da revista:
GENETICA
Ano de publicação:
2011
Tipo de documento:
Article
País de afiliação:
Alemanha