Enrichment for gene targeting in mammalian cells by inhibition of poly(ADP-ribosylation).
Biochim Biophys Acta
; 1308(3): 241-50, 1996 Sep 11.
Article
en En
| MEDLINE
| ID: mdl-8809116
Inhibition of poly(ADP-ribosylation) reduces random genomic integration of transfected DNA and mildly stimulates intrachromosomal homologous recombination in mammalian cells. We investigated the effect of inhibition of poly(ADP-ribosylation) on the efficiency of gene targeting in Chinese hamster ovary (CHO) cell line ATS-49tg. This cell line is hemizygous for a defective adenine phosphoribosyltransferase (aprt) gene and is hypoxanthine phosphoribosyltransferase (hprt) deficient. Plasmid pAG100 contains a portion of the CHO aprt gene sufficient to correct the defect in ATS-49tg cells via gene targeting; pAG100 also contains an Escherichia coli guanine phosphoribosyltransferase (gpt) gene. Following transfection of ATS-49tg cells with pAG100, selection for gpt-positive transfectants allowed recovery of cells that had randomly integrated pAG100 while selection for aprt-positive cells allowed recovery of cells that had undergone gene targeting at the endogenous aprt locus. Treatment of cells with 3 mM 3-methoxybenzamide (3-MB), an inhibitor of poly(ADP-ribose) polymerase, decreased random integration and gene targeting of electroporated pAG100 about 5-fold. In contrast, treatment with 3 mM 3-MB during calcium phosphate transfection could reduce random integration more than 150-fold while reducing gene targeting less than two-fold. Therefore, as much as a 100-fold enrichment for gene targeting was achieved with calcium phosphate transfection.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Poli Adenosina Difosfato Ribosa
/
Transfección
/
Marcación de Gen
/
Inhibidores de Poli(ADP-Ribosa) Polimerasas
Límite:
Animals
Idioma:
En
Revista:
Biochim Biophys Acta
Año:
1996
Tipo del documento:
Article
País de afiliación:
Estados Unidos
Pais de publicación:
Países Bajos