Retrofitting BACs with G418 resistance, luciferase, and oriP and EBNA-1 – new vectors for in vitro and in vivo delivery
Retrofitting BACs with G418 resistance, luciferase, and oriP and EBNA-1 – new vectors for in vitro and in vivo delivery
复制标题
用 G418 抗性、荧光素酶、oriP 和 EBNA-1 改造 BAC——用于体外和体内递送的新载体
作者：
Christine Magin;G. Kotzamanis;L. D’Aiuto;E. Wagner;C. Huxley
背景细菌人工染色体（Bacterial artificial chromosomes，BAC）已广泛用于人类和小鼠基因组测序，因此可用于大多数基因的测序。BAC的大尺寸意味着它们通常可以携带完整的基因，这些基因具有驱动组织特异性表达的全部水平的长距离控制元件。对于基因表达的研究和基因治疗的应用，它是有用的，能够改造的BAC与选择性基因，如G418电阻，报告基因，如荧光素酶，和oriP/EBNA-1从爱泼斯坦巴尔病毒，允许长期的附加型维护在哺乳动物celles.ResultsWe描述了一系列的改造质粒和协议，在体内loxP/Cre重组。载体pRetroNeo携带G418抗性盒，pRetroNeoLuc携带G418抗性和荧光素酶表达盒，pRetroNeoLucOE携带G418抗性、荧光素酶和oriP/EBNA-1盒，pRetroNeoOE携带G418抗性和oriP/EBNA-1。这些载体可以有效地改造到BAC上，而不重排BAC克隆。荧光素酶盒在组织培养物中瞬时转染B16 F10细胞后和在体内电穿孔到BALB/c小鼠的肌肉中后从改造质粒和改造的BAC有效表达。我们还表明，携带GFP、oriP和EBNA-1的BAC可以用Lipofectamine 2000转染到B16 F10细胞中，并可以在5周后完整地拯救。结论pRetro载体允许使用Cre的体内表达有效改造具有G418抗性、荧光素酶和/或oriP/EBNA-1的BAC。荧光素酶报告基因在将改造的BAC瞬时转染到组织培养物中的细胞中后以及在体内电穿孔到小鼠肌肉中后表达。OriP/EBNA-1允许稳定维持150-kb BAC而不重排至少5周。
BackgroundBacterial artificial chromosomes (BACs) have been used extensively for sequencing the human and mouse genomes and are thus readily available for most genes. The large size of BACs means that they can generally carry intact genes with all the long range controlling elements that drive full levels of tissue-specific expression. For gene expression studies and gene therapy applications it is useful to be able to retrofit the BACs with selectable genes such as G418 resistance, reporter genes such as luciferase, and oriP/EBNA-1 from Epstein Barr virus which allows long term episomal maintenance in mammalian cells.ResultsWe describe a series of retrofitting plasmids and a protocol for in vivo loxP/Cre recombination. The vector pRetroNeo carries a G418 resistance cassette, pRetroNeoLuc carries G418 resistance and a luciferase expression cassette, pRetroNeoLucOE carries G418 resistance, luciferase and an oriP/EBNA-1 cassette and pRetroNeoOE carries G418 resistance and oriP/EBNA-1. These vectors can be efficiently retrofitted onto BACs without rearrangement of the BAC clone. The luciferase cassette is expressed efficiently from the retrofitting plasmids and from retrofitted BACs after transient transfection of B16F10 cells in tissue culture and after electroporation into muscles of BALB/c mice in vivo. We also show that a BAC carrying GFP, oriP and EBNA-1 can be transfected into B16F10 cells with Lipofectamine 2000 and can be rescued intact after 5 weeks.ConclusionThe pRetro vectors allow efficient retrofitting of BACs with G418 resistance, luciferase and/or oriP/EBNA-1 using in vivo expression of Cre. The luciferase reporter gene is expressed after transient transfection of retrofitted BACs into cells in tissue culture and after electroporation into mouse muscle in vivo. OriP/EBNA-1 allows stable maintenance of a 150-kb BAC without rearrangement for at least 5 weeks.
影响因子：
7
作者：
Wang, ZD;Engler, P;Storb, U
通讯作者：
Storb, U
影响因子：
3.5
作者：
METCALF, WW;WANNER, BL
通讯作者：
WANNER, BL