Abstract
A novel shuttle vector, pBH140, has been constructed that allows stable maintenance of large genomic inserts as human artificial episomal chromosomes (HAECs) in mammalian cells. The vector, essentially a hybrid BAC–HAEC, contains an F-based replication system as in a bacterial artificial chromosome (BAC) and the Epstein–Barr virus (EBV) latent origin of replication system, oriP, for replication in human cells. A 185-kb DNA insert containing the entire human β-globin locus, including its locus control region (LCR), was retrofitted into this vector. The resulting β-globin BAC–HAEC clone, p148BH, was transfected into human cells and analyzed for episomal maintenance and expression of the β-globin gene. FISH revealed an association of the vector with different human chromosomes but no integration. The β-globin BAC–HAECs were present at an average copy number of 11–15 per nucleus in the stably transformed human cells. After 1 year of continuous in vitro cultivation, the HAECs persisted as structurally intact 200-kb episomes. While no β-globin transcription could be detected in the parental D98/Raji cells, correctly spliced RT-PCR products were produced at significant levels in long-term cultures of the BAC–HAEC-transduced cells. The wide availability of BAC and PAC libraries, the ease in manipulating cloned DNA in bacteria, and the episomal stability of the pBH140 vector make this system ideal for studies on gene expression and other genomic functions in human cells. The potential significance of large, functionally active episomes for gene therapy is discussed.
Overview summary
In this article, a second-generation hybrid BAC–HAEC vector pBH140 has been constructed for shuttling large human inserts from preexisting bacterial libraries such as P1, PAC, and BAC into human cells, following simple and standard cloning techniques. This vector has a large cloning capacity due to its BAC-based backbone and a multiple cloning site with rare cutter restriction sites. The presence of the EBV replication origin oriP and expression of its viral transactivator EBNA-1 on pBH140 allows shuttling of large bacterial episomes into human cells. Using this vector, we report the establishment and maintenance of 200-kb human genomic inserts as stable episomes in EBNA-1-positive human cells for more than 1 year. Using the human β-globin multigene cluster as a model, ectopic and persistent transcription of the human β-globin gene on the extrachromosomal large genomic elements was observed over such an extended cultivation period.
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