Progress Toward the Culture and Transformation of Chicken
YOULIANG WANG, CARRIE F. BROOKS, STEVEN A. JONES, LYN K. OLLIFF, MICHAEL MORGAN,
GORDON L. SPEKSNIJDER, CHUCK FOLEY, ALEX J. HARVEY
AviGenics, Inc., Georgia BioBusiness Center, Athens, Georgia, USA
Key Words. Avian transgenesis • Blastodermal cell culture • Embryonic stem cells
Chicken blastodermal cells can be cultured for short
periods of time and retain the ability to contribute to
somatic and germline tissues when injected into ?-irradi-
ated stage X embryos. Such a method has yet to yield a
germline transgenic bird, in part due to the low rate of
transgene integration into the avian genome. In addition,
the short culture period precludes the identification and
expansion of those cells that carry an integrated trans-
gene. In this study, two methods were developed that
produced blastodermal cells isolated from stage X Barred
Plymouth Rock embryos bearing an integrated transgene.
Addition of chick embryo extract to the culture medium
enabled expansion of single colonies for multiple passages.
Southern blot analysis indicated that the transgenes had
integrated as a single copy in most of the clones. Cells
from passaged, transgenic embryo cells were injected into
irradiated stage X White Leghorn embryos, producing
hatched chicks that bore the donor cells in their somatic
tissues. Transgene sequences were detected in sperm
DNA; however, breeding of chimeras did not result in
germline transmission of the transgene, indicating that
the contribution of the transgenic cells to the germline
was either nonexistent or very low. STEM CELLS 2006;24:
Blastodermal cells (BDCs) isolated from stage X embryos have
been an attractive vehicle for avian transgenesis, yet there re-
main major hurdles that prevent the production of transgenic
flocks of chickens. BDCs are readily isolated from freshly
oviposited eggs, making them amenable to collection and direct
injection into recipient embryos. Each embryo can yield tens of
thousands of cells, which can be transfected by numerous tech-
niques. Most importantly, the cells can contribute to somatic and
germline tissues upon reintroduction into recipient embryos .
Even with all of these advantages, however, the introduction of
DNA into manipulated BDCs has yet to produce a transgenic
chicken that is able to pass the transgene through the germline.
This stems from two major hurdles: the low rate at which
exogenous DNAs integrate into the genome and the propensity
of BDCs cultured for any significant period of time to lose the
ability to contribute to germline tissues.
Injection of Barred Plymouth Rock (BPR) BDCs into a
White Leghorn (WL) embryo results in chicks that bear patches
of black feathers derived from the BPR cells that incorporated
into the WL embryo. Donor BPR cells can also contribute to
germ tissues, which can be determined by breeding of the
chimeric roosters to BPR hens . Irradiation of recipient WL
embryos prior to injection significantly increases the proportion
of chimeric tissue that originated from injected cells such that
when chimeras are bred, up to 100% of their offspring are
donor-derived chicks [1, 2]. Hatchability of the injected egg is
as high as 60% due to improvements in the windowing meth-
odologies [3, 4].
BDCs are readily transfected, typically by addition of
cationic liposome/DNA complexes to the cells in culture
[5–7] or by electroporation [8, 9]. To generate birds that carry
genetically modified BDCs, attempts have been made to
enrich for BDCs that harbor the transgene in their nuclei
either by fluorescence-activated cell sorting (FACS) of cells
transfected with a reporter gene that could be tracked with a
fluorescent dye  or enrichment by application of a mag-
netic field . It was conclusively shown through tracking of
the BPR black feather allele that FACS-sorted cells could
efficiently contribute to somatic and germline tissues .
However, there was no evidence of persistence of the trans-
gene in hatched chicks. Magnetically sorted cells were also
able to contribute the somatic tissues, based on expression of
the lacZ gene in embryos, although no chicks were hatched in
this study, so it was difficult to assess whether the transgene
had stably integrated.
Correspondence: Alex J. Harvey, Ph.D., AviGenics, Inc., Georgia BioBusiness Center, 111 Riverbend Road, Athens, Georgia 30605,
USA. Telephone: 706-227-1170; Fax: 706-227-2180; e-mail: firstname.lastname@example.org
publication March 20, 2006. ©AlphaMed Press 1066-5099/2006/$20.00/0 doi: 10.1634/stemcells.2005-0491
Received October 4, 2005; accepted for
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