Retrofitting BACs with G418 resistance, luciferase, and oriP and EBNA-1 - new vectors for in vitro and in vivo delivery.
BACKGROUND
Bacterial 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.


RESULTS
We 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.


CONCLUSIONS
The 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.
Magin-Lachmann
Christine
C
Boehringer Ingelheim Austria GmbH, A-1121 Vienna, Austria. christine_magin_lachmann@baxter.com
Kotzamanis
George
G
D'Aiuto
Leonardo
L
Wagner
Ernst
E
Huxley
Clare
C
D000818:Animals; D001709:Biotechnology / Q000379:methods*; D045744:Cell Line, Tumor; D022202:Chromosomes, Artificial, Bacterial; D004587:Electrophoresis, Agar Gel; D018274:Electroporation; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D015316:Genetic Therapy; D005822:Genetic Vectors; D005839:Gentamicins / Q000494:pharmacology; D066298:In Vitro Techniques; D008156:Luciferases / Q000378:metabolism*; D008546:Melanoma, Experimental; D051379:Mice; D008807:Mice, Inbred BALB C; D008957:Models, Genetic; D009132:Muscles / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D019309:Epstein-Barr Virus Nuclear Antigens; D005839:Gentamicins; C010680:antibiotic G 418; D008156:Luciferases; C417029:EBV-encoded nuclear antigen 1