* Home

* STC Project History

* 1995 Meeting

* Articles

* Contacts

* Links

* HGP Sequences

* HGP Research

* What is a BAC?

Human Artificial Episomal Chromosome (HAECS) for Cloning, Shuttling and Functional Assay of Large Genetic Units in Human and Rodent Cells*

Min Wang, Panayotis A. Ioannou**, Michael Grosz, Subrata Banerjee, Evy Bashiardes**, Michelle Rider, Tian-Qiang Sun*** and Jean-Michel H. Vos***

Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC [Vos: 919 966-3036 (Phone); 919 966-3015 (Fax) and vos@tmed.unc.edu (E-mail)].

Of some 100,000 human genes, only a few thousand have been cloned, mapped or sequenced so far. Much less is known about other chromosomal regions such as those involved in DNA replication, chromatin packaging, and chromosome segregation. Construction of detailed physical maps is only the first step in localizing, identifying and determining the function of genetic units in human cells. Studying human gene function and regulation of other critical genomic regions that span hundreds of kilobase pairs of DNA requires the ability to clone an entire functional unit as a single DNA fragment and transfer it stably into human cells.

We have developed a human artificial episomal chromosome (HAEC) system based on latent replication origin of the large herpes Epstein-Barr virus (EBV) for the propagation and stable maintenance of DNA as circular minichromosomes in human cells.[1,2] Individual HAECS carried human genomic inserts ranging from 60 to 330 kb and appeared genetically stable. An HAEC library of 1500 independent clones carrying random human genomic fragments with average sizes of 150 to 200 kb was established and allowed recovery of the HAEC DNA. This autologous HAEC system with human DNA segments directly cloned in human cells provides an important tool for functional study of large mammalian DNA regions and gene therapy.[3,4]

Current efforts are focused on (a) shuttling large BAC/PAC genomic inserts in human and rodent cells and (b) packaging BAC/PAC/HAEC clones as large infectious Herpes Viruses for shuttling genomic inserts between mammalian cells and (c) constructing bacterial-based human and rodent HAEC libraries. (a) We have designed a "pop-in" vector, which can be inserted into current BAC-or PAC-based clone via site-specific integration. This "CRE-LOXP"-mediated system has been used to establish BAC/PAC up to 250 kb in size in human cells as HAECS. (b) We have obtained packaging of 160-180 kb exogenous DNA into infectious virions using the human lymphotropic Epstein-Barr virus. After delivery into human beta-lymphoblasts cells the HAEC DNA was stabley established as 160-180 kb functional autonomously replicating episomes.[5,7] We have also generated a hybrid BAC/HAEC vector, which can shuttle large DNA inserts, i.e., at least up to 260 kb, between bacteria and human cells. Such a system is being used to develop large insert libraries, whose clones can be directly transferred into human or rodent cells for functional analysis. These HAEC-derived systems will provide useful molecular tools to study large genetic units in humans and rodents, and complement the functional interpretation of current sequencing efforts.

* Supported by the Office of Health and Environmental Research, Human Genome Program, Department of Energy, under Contract No. DE-FG05-91ER61135

** The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus.

*** Department of Biochemistry and Biophysics; University of North Carolina, Chapel Hill, North Carolina 27599.

[1] Sun, T.-Q., Fenstermacher, D. & Vos, J.-M.H. Human artificial episomal chromosomes for cloning large DNA in human cells Nature Genet 8, 33-41 (1994).

[2] Sun, T.-Q. & Vos, J.-M.H. Engineering of 100-300 kb of DNA as persisting extrachromosomal elements in human cells using the HAEC system in Methods molec. Genet. (ed. Adolph, K.W.) (Academic Press, San Diego, CA, 1995).

[3] Vos, J.-M.H. Herpesviruses as Genetic Vectors in Viruses in Human Gene Therapy (ed. Vos, J.-M.H.) 109-140 (Carolina Academic Press & Chapman & Hall, Durham N.C., USA & London, UK, 1995).

[4] Kelleher, Z. & Vos, J.-M. Long-Term Episomal Gene Delivery in Human Lymphoid Cells using Human and Avian Adenoviral-assisted Transfection. Biotechniques 17, 1110-1117 (1994).

[5] Banerjee, S., Livanos, E. & Vos, J.-M.H. Therapeutic Gene Delivery in Human beta-lymphocytes with Engineered Epstein-Barr Virus. Nature Medicine, Accepted

[6] Sun, T.-Q., Livanos, E., & Vos, J.-M.H. Infectious HAECS for Disease Correction. Nature Medicine, Submitted.

[7] Wang, S. & Vos, J.-M.H. An HSV/EBV based vector for High Efficient Gene Transfer to Human Cells in vitro/in vivo. Submitted


Abstracts scanned from text submitted for January 1996 DOE Human Genome Program Contractor-Grantee Workshop.

Last modified: Wednesday, October 22, 2003

Disclaimers * Webmaster

Home * Links * Meetings * Articles * Contacts * History