A de novo SAC was constructed by making use of YAC technology and a humanized yeast strain. The construct (about 310 kb) contained pig centromeric DNA and the Neo gene. The construct was introduced into a pig cell line by yeast-mammalian cell fusion and 6418 resistant clones were obtained. One clone was characterized by FISH and shown to contain an episomally located microchromosome containing YAC, Neo and pig centromere sequences. FISH analysis over time showed that the SAC was mitotically stable for at least 34 generations in the absence of selection. The size of the SAC was determined by confocal microscopy of the SAC and shown to be approximately 7 Mb; which is about 25-fold greater than the size of the original YAC. From its behavior. in pulsed field gel electrophoresis, FISH analysis of stretched DNA fibers, and its appearance under scanning confocal microscopy, it was concluded that the SAC is a circularized and multimerized derivative of the original YAC. Possible applications as vectors for animal transgenesis are discussed. (C) 2002 Editions scientifiques et medicales Elsevier SAS and Societe francaise de biochimie et biologie moleculaire. All rights reserved.
Poggiali, P., Scoarughi, G., Lavitrano, M., Donini, P., Cimmino, C. (2002). Construction of a swine artificial chromosome: a novel vector for transgenesis in the pig. BIOCHIMIE, 84(11), 1143-1150 [10.1016/S0300-9084(02)00019-6].
Construction of a swine artificial chromosome: a novel vector for transgenesis in the pig
LAVITRANO, MARIALUISA;
2002
Abstract
A de novo SAC was constructed by making use of YAC technology and a humanized yeast strain. The construct (about 310 kb) contained pig centromeric DNA and the Neo gene. The construct was introduced into a pig cell line by yeast-mammalian cell fusion and 6418 resistant clones were obtained. One clone was characterized by FISH and shown to contain an episomally located microchromosome containing YAC, Neo and pig centromere sequences. FISH analysis over time showed that the SAC was mitotically stable for at least 34 generations in the absence of selection. The size of the SAC was determined by confocal microscopy of the SAC and shown to be approximately 7 Mb; which is about 25-fold greater than the size of the original YAC. From its behavior. in pulsed field gel electrophoresis, FISH analysis of stretched DNA fibers, and its appearance under scanning confocal microscopy, it was concluded that the SAC is a circularized and multimerized derivative of the original YAC. Possible applications as vectors for animal transgenesis are discussed. (C) 2002 Editions scientifiques et medicales Elsevier SAS and Societe francaise de biochimie et biologie moleculaire. All rights reserved.
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