Conventional gene targeting protocols lead to loss of targeted cells when applied to a silent gene locus in primary fibroblasts.
J Biotechnol
; 125(2): 185-93, 2006 Sep 01.
Article
em En
| MEDLINE
| ID: mdl-16621085
ABSTRACT
Gene targeting in livestock fibroblasts has proven difficult to achieve, particularly if the target gene is silent. We first tested whether efficient gene targeting at the transcriptionally active ovine alpha1(I) procollagen (COL1A1) locus required the use of a promoter trap vector. We compared gene targeting frequencies at the ovine COL1A1 locus using both a promoter trap and a non-promoter trap selection strategy. We demonstrated that targeted cells could be isolated regardless of whether an enrichment step (promoter trap) was used. Next, we used our optimised protocol to target a non-expressed gene, ovine beta-casein. We obtained clones that were scored positive by PCR for the targeting event, but were negative after cell expansion and Southern analysis. We propose that targeted cells were initially generated but that they were at a selective growth disadvantage during culture. We suggest modifications to the conventional targeting protocol that would prevent such loss of targeted cells.
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Bases de dados:
MEDLINE
Assunto principal:
Marcação de Genes
/
Fibroblastos
Tipo de estudo:
Prognostic_studies
Limite:
Animals
Idioma:
En
Revista:
J Biotechnol
Assunto da revista:
BIOTECNOLOGIA
Ano de publicação:
2006
Tipo de documento:
Article
País de afiliação:
Espanha