Input DNA ratio determines copy number of the 33 kb Factor IX gene on de novo human artificial chromosomes.
Human artificial chromosomes (ACs) are non-integrating vectors that may be useful for gene therapy. They assemble in cultured cells following transfection of human centromeric alpha -satellite DNA and segregate efficiently alongside the host genome. In the present study, a 33 kilobase (kb) Factor IX (FIX) gene was incorporated into mitotically stable ACs in human HT1080 lung derived cells using co-transfection of a bacterial artificial chromosome (BAC) harboring synthetic alpha -satellite DNA and a P1 artificial chromosome(PAC) that spans the FIX locus. ACs were detected in >or=90% of chromosome spreads in 8 of 19 lines expanded from drug resistant colonies. FIX transgene copy number on ACs was determined by input DNA transfection ratios. Furthermore, a low level of FIX transcription was detected from ACs with multiple transgenes but not from those incorporating a single transgene, suggesting that reducing transgene number may limit misexpression. Their potential to segregate cross species was measured by transferring ACs into mouse and hamster cell lines using microcell-mediated chromosome transfer. Lines were obtained where ACs segregated efficiently. The stable segregation of ACs in rodent cells suggests that it should be possible to develop animal models to test the capacity of ACs to rescue FIX deficiency.
Department of Medical and Molecular Genetics, Indiana University School of Medicine, 975 W. Walnut Street, IB130, Indianapolis, Indiana, USA.
Amy M
AM
Breman
Camie M
CM
Steiner
Roger B
RB
Slee
Brenda R
BR
Grimes
D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D002478:Cells, Cultured; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D022222:Chromosomes, Artificial, Human / Q000235:genetics*; D006224:Cricetinae; D003412:Cricetulus; D005164:Factor IX / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D008957:Models, Genetic; D016133:Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000235:genetics; D014162:Transfection / Q000379:methods; D019076:Transgenes / Q000235:genetics
D005164:Factor IX