Generation of a telomere-based episomal vector.
We have developed a telomere-based episome by large-scale amplification in Escherichia coli cells. This episome consists of a PAC vector in which a 6 Kb sequence, containing an array of telomeric repeats spaced by a synthetic sequence, is tandemly repeated by large-scale multimerization in E. coli. After transfection in human HT1080 cells, the construct, called clone 106, was able to persist in episomal form or integrated into some endogenous chromosomes. Integrations occurred exclusively at the telomeres. Episomes were still present in HT1080 cells after more than 100 days in the absence of selection. Integrations of clone 106 into the telomeric regions were retained only under selective conditions, and when the selection was removed the construct was progressively eliminated from the chromosome. The long-term maintenance of clone 106 into human cells as an episome and its ability to integrate transiently into the telomeres of the host chromosomes suggest that this PAC-based episome is potentially a good candidate vector for gene therapy applications.
D'Aiuto
Leonardo
L
Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15219, USA. daiutol@msx.upmc.edu
de las Heras
Jose I
JI
Ross
Andrew
A
Shen
Ming Hong
MH
Cooke
Howard
H
D045744:Cell Line, Tumor; D027042:Chromosomes, Artificial, P1 Bacteriophage / Q000235:genetics* / Q000648:ultrastructure; D003001:Cloning, Molecular / Q000379:methods*; D005354:Fibrosarcoma / Q000235:genetics* / Q000473:pathology; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D010957:Plasmids / Q000235:genetics* / Q000648:ultrastructure; D016615:Telomere / Q000235:genetics* / Q000648:ultrastructure; D014162:Transfection / Q000379:methods*