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ATC Workshop Papers
From Cell to Production
Technical Challenges of Cloning Pigs for BioMedical
Research
Somatic Cell Nuclear Transfer in Mammals
SATACs and Transgenesis
Concerns About Gene Transfer and Nuclear Transfer
in Domestic Animals
Prospects and Hurdles in Optimizing the Vascular
Support of Engineered Tissues
ES Cells Make Neurons in a Dish
Nuclear Transfer and Gene Targeting in Domestic
Animals: Bioreactors of the Future
Application of Nuclear Transfer Technology
in the Generation of Pigs for Xenetransplantation
Genomics: Delivering Cell Culture Systems
for Tissue Therapy
Nuclear Transfer Technology
Gene Targeting in Domestic Species: Challenges
and Opportunities
Homologous Recombination and Genetic Engineering
of Transgenic Recombinant Animals
Nuclear Transplantation in the Cow: Future
Challenges
Enhancing Transgenics through Cloning
ES Cells Offer is a Power Tool for Understanding
the Genetic Control of Tissue Development and for Screening Potential
Therapeutic Drugs
Human Germline Engineering -- The Prospects
for Commercial Development
Understanding Developmental Abnormalities
in Offspring Produced by Nuclear Transplantation
Role of Cell Cycle
Cloning and Other Reproductive Technologies
for Application in Transgenics
Cell Culturing Technology as a Major Hurdle
in the Commercialization of Genetically Altered Animals
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| ADVANCED TRANSGENESIS AND
CLONING: Genetic Manipulation in Animals
Electronic Workshop Presentation: Paper
No. 19
MAMMALIAN ARTIFICIAL CHROMOSOMES
FOR ANIMAL TRANSGENESIS
Participant:
Jean-Michel H. Vos, D.Sc.
Principale Investigator
Associate Professor of Biochemistry,
Genetics, & Molecular Biology
Lineberger Comprehensive Cancer Center
University of North Carolina, Chapel Hill, NC 27599-7295
Overview
With the recent advances on whole animal cloning, one can expect that
transgenic animals will be soon generated by merging such technology
with powerful molecular and genetic tools. There are 2 general strategies
for engineering transgenic animals of commerical interest: A) transgene
insertion at pre-selected chromosomal sites, and B) transgene inclusion
on mammalian artificial chromosomes (MAC). Since the former experimental
approach is the topic of discussion by other speakers from this forum,
the focus of this presentation will be on the later one.
Potential Technological Benefits of MACs
Before summarizing the various potential MAC technologies, the
following list outlines some of the fundamental aspects in developing
MAC-based animal transgenesis for commercial purpouses: a) Transgene
expression on MACs eliminates position effects associated with chromosomal
insertion. b) MAC-based transgenes can be handled as large intact
genomic fragments spanning critical genetic elements for adequate
and regulated expression. c) The opportunity for DNA rearrangement
frequently observed with chromosomal integration can be minimized
with MACs. d) The usage of MACs reduces the risks of mutational
insertion of the host genome by transgenic sequences. e) Disabling
features can be included as safety precaution (i.e. conditional
"suicide" MACs), a process difficult to accomplish with stably integrated
transgenes.
Trimming-down Natural Chromosomes
As illustrated in Figure 1, two general strategies can be envisioned
for creating MAC-based transgenic animals expressing transgenes
of commercial values: a) in situ engineering by introducing the
individual MAC components into the cell types currently used for
generating transgenic animal, i.e. zygotes and embryonal stem cells;
or b) alternatively, delivery of pre-assembled MACs from "parental"
donor cells (i.e. somatic mammalian cell lines or microbial cells)
into "target" recipient zygotic or ES cells. Because the first experimental
strategy allows less control on the building process leading to
a functional MAC, the second general approach appears more reliable
and reproducible at the long-term. The construction of MACs can
be performed following a "top-down" or "bottom-up" experimental
scheme (Fig. 1). The top-down strategy relies on the progressive
fragmentation of natural mammalian chromosomes by repetitive targeted
deletions. Potential drawbacks of this approach are i) inherent
genomic instability and resistance to extreme size reduction; ii)
inability to manipulate the MAC DNA in vitro and hence difficulty
in large-scale production; and iii) inefficient transgene insertion
into such large MACs and their subsequent transfer into the zygotic
(or ES) cells.
Click here to view large
scale version of figure.
Assembly in "Incubator" Cell Lines
In contrast, the bottom-up strategy relies on the modular incorporation
of the minimal genetic cis-elements necessary and sufficient for
generating a functional MAC, i.e. autonomous replication, mitotic
(and meiotic) segregation and genomic stability. Two general bottom-up
approaches (Fig. 1) are currently being developed based on, respectively,
a) the in situ assembly of the MAC by co-delivery of the various
DNA components into appropriate "incubator" mammalian cell lines,
or b) the in vitro pre-assembly of the MAC using microbial systems,
i.e. bacteria or yeast. a) Current drawbacks of the in situ methodology
are: i) inefficient and uncontrolled joining of the various transfected
DNA components by the cellular machinery; and ii) large size of
the MACs rendering difficult their mass production, genetic manipulation
and shuttling into zygotic (or ES) target cells.
Pre-assembly Using Microbial Systems
By analogy to large cloning systems in micro-organisms such as
the bacterium Escheriachia coli and budding yeast Saccharomyces
cerevisiae, MACs can be constructed in such microbial systems using
endogenous chromosomal elements from mammalian genomes such as the
yeast-based YACs (Yeast Artificial Chromosomes), or exogenous extra-chromosomal
elements derived from viruses and other mammalian parasites such
as the bacterial-based BACs (Bacterial Artificial Chromosomes) and
PACs (P1 Artificial Chromosomes). Because of the improved control
on the various assembly phases for building MACs, an in vitro approach
is expected to reduce the various problems outlined above in the
in situ section. Technical difficulties in manipulating, purifying
and transferring linear (or circular) YACs from yeast into mammalian
cells led the author's research group to focus its efforts on developing
technologies for the contruction of large stable circular MACs which
relyied on a combination of bacterial, viral and genomic systems.
Specifically, the author's approach has been to merge endogeneous
chromosomal elements with exogeneous extra-chromosomal ones. Below
are summarized the main steps and technological features of such
a "chimeric MACs" technology at its current stage of development
(Figure 2).
Click here to view large
scale version of figure.
Human Artifical Episomal Chromosomes (HAECs & BAC-HAECs)
The author's long-term goal has been to build large (i.e. 100-500kb)
artificial circular mammalian episomes capable of autonomous replication
and proper segregation in situ (i.e. cultured cells) and in vivo (gene
therapy and animal transgenesis). Such a size range appears sufficient
for spanning most mammalian genes as a single MAC and the circular
structure is also more appropriate for the in vitro manipulation of
the MAC. In the first phase, the following strategy was devised using
human cells. To establish the large circular self-replicating MACs
in human cells, non-infectious and infectious episomal vectors based
on the latent human herpes Epstein-Barr virus (EBV) were developed.
A first-generation HAEC vector was engineered for the in vitro assembly
and shuttling into human cells of episomes carrying large DNA fragments
(i.e. HAEC library).
In the next phase, a two-step strategy using a second-generation
BAC-HAEC vector was developed based on i) the contruction of large
artifical circular episomes in bacteria and ii) their transfer into
target mammalian cells (Figure 2). This system can shuttle large
DNA clones from pre-existing bacterial (BAC, P1, PAC) or yeast (YAC)
libraries into cultured cells following simple and standard techniques.
As illustration, our laboratory has used this system for the engineering,
transfer, stable maintenance and expression for more than one year
of human genes carried as 200 kb episomes in human cells. The wide
availability of BAC and PAC libraries, the ease in manipulating
cloned DNA in bacteria, and the episomal stability of this novel
BAC-HAEC vector make this technology ideal for the pre-assembly
of MACs in bacteria, followed by their transfer into target mammalian
cells.
Mouse Artifical Episomal Chromosomes (MAECs)
The availability of an in vitro assembly system working in different
mammalian cell species would enable in vivo testing of MACs in various
live-stock animals. As a proof-of-concept, a strategy was developed
for the shuttling of 100-200 kb circular human-based episomes into
rodent cells. Using microcell fusion and more recently DNA transfection
as methods for inter-species cell shuttling, Mouse Artificial Episomal
Chromosomes (MAECs) carrying 95-105 kb of human DNA (HAECs & BAC-HAECs)
have been established in mouse cells. Such MAECs were stably maintained
for at least half-a-year with a 95% episomal retention per cell division.
The establishment of such a first-generation MAEC system should facilitate
the transfer of MACs pre-assembled in bacteria into other mammalian
cell types, and allow to study the genetic components required for
shuttling and maintenance of large circular MACs in various live-stock
mammals.
Transgenic Episomal Artificial Mammals (TEAM)
The ability to establish large artificial circular episomes replicating
stably in mouse cells allows to initiate experiments with whole animals.
As a proof-of-concept, a murine-based transgenic strategy was developed
based on the results from the above in vitro experiments. Using HAECs
and MAECs isolated from human and mouse cells respectively, over 100
transgenic mice carrying large circular episomes have been generated
over the last year, i.e. transgenic episomal artificial mammals (TEAM).
In particular, this experimental approach was characterized by an
unusually high frequency of transgenesis (i.e. 50% or more) and persistence
of the episomes over time (i.e. 8 months or more). Altogether, the
above results suggest that the combined HAEC, BAC-HAEC, MAEC and TEAM
technologies have potentials for engineering stable MACs functioning
effectively in different transgenic animals.
Prospects on the Author's Work
Current efforts in the author's laboratory are focused on several
areas, including: i) development of artificial circular episomes working
in cells from various live-stock species ; ii) identification of cis-elements
from different mammalian genomes controlling long-term MAC persistence;
iii) testing therapeutic MACs with complementing disease transgenes
in knock-off mice models; iv) comparing nonviral and viral-based systems
for the delivery of pre-assembled MACs into zygotic and embryonal
stem cells. It is the author's hope that the above oultined plan for
engineering in vitro pre-assembled circular MACs using the TEAM system
will help drive the technology of animal transgenesis and cloning
effectively and competitively into the new millenium.
Commercial and Technological Perpectives
The development of MACs is expected to be useful in many areas
of biotechnological and biomedical endeavours, including animal
transgenesis, functional genomics and gene therapy. To reach such
a stage of applications, several technical hurdles will have to
be circumvented. For example, most current technologies are restricted
by the inefficiency and delivery constraints of much too large MACs.
In addition, the effect of poorly understood epigenetic phenomena
for de novo replication and segregation activities of MACs will
have to be critically analyzed. Once these problems are solved,
commercial applications will be numerous, particularly with systems
involving large and/or multiple genes. In conclusion, the biotechnological
industries will greatly benefit from the generation of transgenic
animals carrying modular, pre-assembled, sequence-defined and stably
inherited MACs with regulated transgenes and selected phenotypes
of commercial value.
Author's Related Bibliography
General Reading
J-M Vos (1995) “Herpes Viruses as Genetic Vectors” In
Viruses in Human Gene Therapy, ed. J-M Vos, Carolina Academic Press,
Durham, NC pp 109-140.
J-M. H. Vos, Westphal E.V. and Banerjee S.(1996)“Infectious
Herpesvirus vectors for Gene Therapy” IN Gene Therapy, EDS
N.R. Lemoine and D. Cooper, Bios Scientific Publisher, Oxford, U.K.,
Chapter 8, pp. 127-153.
J-M. H. Vos, “The Simplicity of Complex MACs” Nature
Biotechnology, (1997) 15:1257-1259.
J-M.H. Vos, (1998) “HEACs and MAECs Technologies: Applications
to Functional Genomics of large DNA in Human and Mouse Cells”
Human Genome News, 9(1-2), 6.
J-M.H. Vos, (1998) “MACs as Tools for Gene Therapy” Curr.
Op. in Genet. and Dev., 8:351-359.
In-Depth Reading
T-Q Sun, D. Fenstermacher and J-M Vos “Human Artificial Episomal
Chromosomes for Cloning Large DNA in Human Cells” (1994) Nature
Genetics 8:33-41.
S. Banerjee, E. Livanos, J-M. H. Vos, (1995) “Therapeutic
Gene Delivery in Human lymphocytes with Non-transforming Engineered
Epstein-Barr Virus”. Nat. Med. I:1303-1308.
T. Q. Sun, E. Livanos, J-M. H. Vos. (1996)“Engineering a Mini-Herpesvirus
as a General Strategy to Transduce up to 180kB of Functional Self-Replicating
Human Mini-Chromosomes” Gene Ther. 3: 1081-1088.
S. Wang, J-M. H. Vos (1996)“An HSV/EBV based vector for High
Efficient Gene Transfer to Human Cells in vitro/in vivo” J.
Virol. 70: 8422-8430.
Z. Kelleher, Fu, H, E. Livanos, Wendelburg, B., Gulino, S and J-M.H.Vos,
(1998) “Epstein-Barr virus for shuttling 100kB human DNA in
mouse cells as mouse artificial episomal chromosomes”, Nature
Biotechnology, 16:762-768.
E-M. Westphal, Sierakowska, H., L. Livanos, R. Kole and J-M. Vos,
(1998) “A system for shuttling 200 kb PAC/BAC clones into human
cells: stable extrachromosomal persistence and long-term ectopic
gene activation”, Human Gene Therapy, 9: 1863-1873.
B.J. Wendelburg and J-M.H. Vos. (1998) “An enhanced EBNA1
variant with reduced Gly-Ala domain for long-term episomal maintenance
of oriP-based plasmids in human cells”, Gene Therapy, in press.
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