Researchers at the University of Pennsylvania School of Veterinary
Medicine have demonstrated the potential of a new strategy for genetic
modification of large animals. The method employs a harmless gene therapy
virus that transfers a genetic modification to male reproductive cells,
which is then passed naturally on to offspring.
Ina Dobrinski, associate professor and director of the Center for
Animal Transgenesis and Germ Cell Research at Penn Vet, and her colleagues
introduced adeno-associated virus, AAV, to male germline stem cells in both
goats and mice. The study showed that AAV stably transduced male germ line
stem cells and led to transgene transmission through the male germ line.

The findings, available online in The FASEB Journal and in the
February 2008 print edition, are the first report of transgenesis via germ
cell transplantation in a non-rodent species, a promising approach to germ
line genetic modification. It also demonstrates that germline transduction
and germ cell transplantation in large animals provides an approach that is
potentially less costly than microinjection and cloning, the traditional
methods used to generate transgenic large animal models for biomedical
research.

Researchers used mouse germ cells harvested from experimentally
induced cryptorchid donor testes that were then exposed in vitro to AAV
vectors carrying a green fluorescent protein transgene and transplanted to
germ cell-depleted recipient testes, resulting in colonization of the
recipient testes by transgenic donor cells.

When researchers mated these recipient males with wild-type females,
10 percent of offspring carried the gene originally introduced into the
transplanted germ cells, meaning the genetic modification had been passed
on. To broaden the approach to non-rodent species, AAV-transduced germ cells
from goats were transplanted to recipient males in which endogenous germ
cells had been depleted by fractionated testicular irradiation. Transgenic
germ cells colonized recipient testes and produced transgenic sperm. When
semen was used for in vitro fertilization, 10 percent of embryos were
transgenic.

?Initially, the team used the established germ cell transplantation
model in the mouse to investigate whether AAV-mediated transduction of germ
cells was possible and could result in transgene transmission,? Dobrinski
said. ?To broaden the applicability of the results for different mammalian
species, the approach was then applied to a large animal species in which
germ cell transplantation-mediated transgenesis would provide an important
alternate approach to the generation of transgenic animal models for
biomedical research.?

Currently, somatic cell nuclear transfer or pronuclear injection is
used to generate transgenic animals. These inefficient and difficult methods
also carry a risk of producing offspring with developmental abnormalities.
The use of retroviral or lentiviral vectors has been reported in rodents,
but it requires that animals be handled and maintained under higher
biosafety precautions that render this approach less practical for
transgenesis in large animal species. In contrast, animals exposed to AAV
can be maintained under standard husbandry conditions.

AAV is a dependent virus that carries no disease and causes only a
very mild response from the immune system. Because AAV can infect both
dividing and non-dividing cells and passes its genome, it is considered an
excellent candidate for use in gene therapy.

[www.eurekalert.org]