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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
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
Mammalian Artificial Chromosomes for Animal
Transgenesis
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. 04
SATACs and TRANSGENESIS
Participant:
Jan I. Drayer, MD, PhD
Vice President R&D
Chromos Molecular Systems Inc.
Vancouver, B.C. Canada
The development of transgenic animals is
highly inefficient in the area of gene alteration of embryonic cells
and in the generation of transgenic animals from these cells.
With current methods less than 5% of the host cells integrate the
foreign DNA into their genome. This integration process occurs in
a random fashion, lacking control of gene expression levels. Genome
position effects may lead to transgene silencing, inappropriate
transgene expression, and tissue variegation. At present, transfection
of intact DNA payloads greater than 100 kb is problematic, and the
activity of the foreign gene(s) can only be ascertained once the
founder animal is born, matured, or producing milk. Finally, there
is no assurance with current methods, that the founder animal’s
offspring will inherit the foreign DNA.
These shortcomings are further amplified by the current limitations
in the efficiency of the actual development of the transgenic animal
from gene-altered fertilised embryonic cells or other cells used
in the generation of these animals (including cells used in the
nuclear transfer process or embryonic stem cells).
Chromos’ Satellite DNA Based Artificial Chromosomes (SATACs)
have the potential to enable the production of protein(s) in milk
of transgenic animals based on large genes or several genes, and
to custom-engineer and test the function of gene constructs before
introduction into the target cell of the animal. SATACs are not
expected to be dependent upon the method of introduction (e.g.,
microinjection, nuclear transfer, or techniques using embryonic
stem cells).
There are four potential methods to produce transgenic animals
using SATACs:
- microinjection of SATACs into the pronucleus of fertilised oocytes;
- transfer of nuclei from foetal fibroblasts, germ cells, or other
somatic cells to enucleated oocytes;
- injection of transgenic embryonic stem cells into blastocysts
and breeding the chimeric offspring; or
- direct delivery of SATACs into the blastocyst cavity and breeding
the mosaic offspring.
We have taken many steps in realising our goal to resolve major
hurdles in the efficient and controlled generation of transgenic
animals. SATAC-platforms have been custom engineered to carry multiple
copies of more than 3 different genes as well as SATACs that carry
greater than 100 tandem copies of a 40 kb cosmid encoding a gene
for protein production in transgenic animals, representing a total
genetic payload of 4,000 kb (Figure 1).
Figure 1: FISH image of a Chinese Hamster Ovary cell line
with murine-based SATAC engineered with 100 tandem copies of a 40-kb
cosmid representing 4,000 kb payload (green).
Currently, Chromos is able to isolate more than 60,000,000 SATACs/week,
at a purity of 99% and a shelf life of two weeks. These 1-micron
by 2-micron SATACs have been introduced into embryos (Figure 2)
by microinjection, resulting in SATAC-containing mosaic avian, murine
and bovine embryos.
Figure 2: Murine oocyte after a pronuclear microinjection
of a Fluorescently-dyed SATAC
However, significant hurdles remain to bring the technology to
the desired efficiencies and predictable outcomes for long term,
stable and regulatable gene delivery and expression in transgenic
animals. These include:
- Optimisation of the microinjection techniques to increase the
number of viable fertilised embryos to > 90%
- Optimisation of the timing of SATAC introduction in the embryonic
cell to achieve true transgenesis
- Development of other delivery technologies to transfer SATACs
to cells used in the transgenesis process, e.g., receptor-based
delivery
- Optimisation of microcell fusion techniques to deliver SATACs
to these cells
- Optimisation of the conditions for effective direct delivery
of SATACs to developing embryos
Resolution of these issues would significantly increase the percentage
of viable transgenic embryos, produce a high rate of founder animals
with predictable fully functional expression of the desired genes
and assure germ-line transmission.
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