Modular bacterial artificial chromosome vectors for transfer of large inserts into mammalian cells

被引:26
作者
Frengen, E
Zhao, BH
Howe, S
Weichenhan, D
Osoegawa, K
Gjernes, E
Jessee, J
Prydz, H
Huxley, C
de Jong, PJ
机构
[1] Univ Oslo, Biotechnol Ctr Oslo, N-0349 Oslo, Norway
[2] Roswell Pk Canc Inst, Dept Canc Genet, Buffalo, NY 14263 USA
[3] Univ London Imperial Coll Sci Technol & Med, London, England
[4] Life Technol, Gaithersburg, MD 20898 USA
关键词
D O I
10.1006/geno.2000.6286
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
To facilitate the use of large-insert bacterial clones for functional analysis, we have constructed new bacterial artificial chromosome vectors, pPAC4 and pBACe4. These vectors contain two genetic elements that enable stable maintenance of the clones in mammalian cells: (1) The Epstein-Barr virus replicon, oriP, is included to ensure stable episomal propagation of the large insert clones upon transfection into mammalian cells. (2) The blasticidin deaminase gene is placed in a eukaryotic expression cassette to enable selection for the desired mammalian clones by using the nucleoside antibiotic blasticidin. Sequences important to select for loxP-specific genome targeting in mammalian chromosomes are also present. In addition, we demonstrate that the attTn7 sequence present on the vectors permits specific addition of selected features to the library clones. Unique sites have also been included in the vector to enable linearization of the large-insert clones, e.g., for optical mapping studies. The pPAC4 vector has been used to generate libraries from the human, mouse, and rat genomes. We believe that clones from these libraries would serve as an important reagent in functional experiments, including the identification or validation of candidate disease genes, by transferring a particular clone containing the relevant wildtype gene into mutant cells or transgenic or knock-out animals. (C) 2000 Academic Press.
引用
收藏
页码:118 / 126
页数:9
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