Ubiquitous GFP expression in transgenic chickens using a lentiviral vector
Susan C. Chapman1,*,
Aaron Lawson4,
William C. MacArthur3,
Russell J. Wiese3,
Robert H. Loechel3,
Maria Burgos-Trinidad3,
John K. Wakefield5,
Ram Ramabhadran5,
Teri Jo Mauch1,2 and
Gary C. Schoenwolf1
1 University of Utah School of Medicine, Department of Neurobiology and Anatomy,
and Children's Health Research Center, Room 401 MREB, 20 North 1900 East Salt
Lake City, Utah 84132-3401, USA
2 University of Utah School of Medicine, Department of Pediatrics, Room 2R063,
20 North 1900 East Salt Lake City, Utah 84132-2204, USA
3 GeneWorks, Avian Transgenics, 3950 Varsity Drive, Ann Arbor, Michigan 48108,
USA
4 University of Ghana Medical School, Accra, Ghana, West Africa
5 Tranzyme Pharma, PO Box 13097, 21 Davis Drive, Research Triangle Park, North
Carolina 27709, USA
*
Author for correspondence (e-mail:
susan.chapman{at}utah.edu)
Accepted 16 December 2004
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SUMMARY
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We report the first ubiquitous green fluorescent protein expression in
chicks using a lentiviral vector approach, with eGFP under the control of the
phosphoglycerol kinase promoter. Several demonstrations of germline
transmission in chicks have been reported previously, using markers that
produce tissue-specific, but not ubiquitous, expression. Using embryos sired
by a heterozygous male, we demonstrate germline transmission in the embryonic
tissue that expresses eGFP uniformly, and that can be used in tissue
transplants and processed by in situ hybridization and immunocytochemistry.
Transgenic tissue is identifiable by both fluorescence microscopy and
immunolabeling, resulting in a permanent marker identifying transgenic cells
following processing of the tissue. Stable integration of the transgene has
allowed breeding of homozygous males and females that will be used to produce
transgenic embryos in 100% of eggs laid upon reaching sexual maturity. These
results demonstrate that a transgenic approach in the chick model system is
viable and useful even though a relatively long generation time is required.
The transgenic chick model will benefit studies on embryonic development, as
well as providing the pharmaceutical industry with an economical
bioreactor.
Key words: GFP, Transgenic, Lentiviral, Chick, Development
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Introduction
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Production of transgenic chicks has been technically challenging, in part,
owing to the nature of the reproductive system of the hen and the processing
of the egg as it passes down the oviduct, the need for the shell in
development, and the difficulty in isolating germ/ES cells. Additional
significant challenges include the requirement for laboratory and animal
housing facilities in close proximity to each other, and the relatively long
generation time required to produce birds of reproductive age. A number of
approaches have been pursued with varying degrees of success: manipulation of
oocytes/spermatozoa or newly laid eggs; production of chimeras using
primordial germ cells or chick ES cell equivalents; and gene transfer using
viral vectors (Mozdziak and Petitte,
2004
; Petitte et al.,
2004
; Sang, 2004
).
Oncogenic viruses have shown promise
(Ishii et al., 2004
),
including avian leucosis virus (ALV) and reticuloendotheliosis virus (REV)
(Salter et al., 1986
;
Salter et al., 1987
;
Bosselman et al., 1989a
;
Bosselman et al., 1989b
), and
Moloney murine leukemia virus (MoMLV)
(Mizuarai et al., 2001
). A
newly reported MoMLV-transduced chick, utilizing the vesicular stomatitis
virus G glycoprotein (VSV-G) vector pseudotyping system, expresses GFP under
control of the rous sarcoma virus (RSV) promoter
(Kwon et al., 2004
). G0 birds
produced using this system are chimeric, but G1 analysis has yet to be
reported.
Recently, lentiviral vectors have been favored, based on their ability to
transduce dividing and non-diving cells
(Naldini et al., 1996
;
Pfeifer et al., 2002
), a
relatively large transgene capacity of 8 kb, and the apparent resistance of
transduced cells to gene silencing, which is a problem with oncogenic viral
vectors. The promoter/enhancer elements driving gene expression are of some
importance, depending on whether ubiquitous or tissue-specific expression is
desired. The cytomegalovirus immediate-early gene promoter/enhancer (CMV) is a
highly efficient promoter in many vertebrates, but in chick it seems to be
less efficient than ß-actin (Colas and
Schoenwolf, 2003
; Krull,
2004
). McGrew et al. (McGrew
et al., 2004
) have reported a CMV-driven GFP transgene in chick
that shows conserved tissue expression in the germline, but not ubiquitous
expression. A newly reported CAGGS enhancer/promoter containing the
ß-actin promoter successfully drives ubiquitous GFP expression in mice,
but awaits full analysis in G1 birds
(Sang, 2004
).
We describe results from embryos expressing GFP ubiquitously under the
control of the phosphoglycerol kinase (PGK) promoter, obtained from eggs
produced by a lentiviral-generated rooster (G2) mated to wild-type hens. The
approach used to generate transgenic chicks provides a model for the
production of a new generation of avian transgenics for investigating the gene
and tissue interactions important in embryonic development. Other applications
include expressing pharmaceutical products in egg albumen
(Harvey et al., 2002a
;
Harvey et al., 2002b
), and
imparting disease resistance to poultry flocks through genetic
manipulation.
 |
Materials and methods
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Viral stocks and injection
A replication-defective HIV-1 lentiviral vector, with packaging vectors
deleted, containing eGFP under the control of the PGK promoter, was obtained
from Tranzyme LLC (RTP, NC). Published guidelines for biosafety level 2 were
followed for injecting the virus. Unincubated fertilized eggs were obtained
from Charles River Laboratories SPAFAS Avian Products and Services (Roanoke,
IL), and were accompanied by a Quality Control Sheet that listed the test
dates and methods used to screen for 28 avian pathogens within the flock. The
eggs were maintained on their sides at room temperature upon arrival and until
microinjection, which happened over the course of two days. When eggs were
kept on their sides, embryos floated to the uppermost point of the shell. Thus
by windowing this area of shell, embryos were exposed directly beneath the
window for subsequent injection.
The upper surface of the egg was cleaned with a solution of 70% ethanol in
water (v/v) and then blotted dry with a laboratory tissue. A small (0.3-cm
diameter) hole was abraded into the shell using a hand-held rotary tool fitted
with an abrasive stone bit. A small hole was then cut through the shell
membrane with a #11 blade disposable scalpel. A glass capillary was attached
to a microinjection apparatus that was attached to a micromanipulator. A
solution of lentivirus, at a titer of 105 to 107
infectious particles/ml, was drawn into the glass capillary that had been
pulled to a tip thickness of a few hundred microns using a Sutter model P-30
pipette puller. The glass capillary was then positioned into the subgerminal
space of the exposed embryo and 2-5 µl of the suspension was delivered.
The space beneath the window in the egg was then filled with PBS containing
penicillin (100 I.U./ml) and streptomycin (100 mg/ml) (Media Tech Cellgro),
and the window was then covered with a donor shell membrane harvested from
eggs grown under sterile conditions and maintained in the PBS/antibiotic
solution described above. After drying in a Class II biological safety
cabinet, the membranes were covered with Opsite® surgical tape and placed
in a Natureform model NMC-4000 Incubator until hatching. Two days before
hatching the eggs were moved to a Natureform model NMC-4000 Hatcher. Eggs were
handled in accordance with standard safety procedures for modified
organisms.
Analysis of transgenic birds
To identify individuals containing the transgene, DNA was extracted from
the blood of hatchlings and from the semen of mature roosters, using a
PUREGENE DNA purification kit (Gentra Systems, Minneapolis, MN), and then
analyzed by PCR. PCR amplifications were performed in a volume of 40 µl,
using PCR supermix (Invitrogen), 2 µl of genomic DNA template, and 0.2
µM of each primer. Primers were as follows:
semen, 5'-ACTCACAGTCTGGGGCATCAAG-3' and
5'-CCACCTTCTTCTTCTAATCCTTCG-3'; and
blood, 5'-GGACAGCAGAGATCCAGTT-3'and
5'-CGGTGGTGCAGATGAACTT-3'.
PCR conditions for both semen and blood were: 94°C for 2 minutes; then
94°C for 45 seconds, 63°C for 10 seconds, cooling at 0.1°C/sec to
62°C, 62°C for 1 minute and 72°C for 1 minute, for 34 cycles;
followed by 72°C for 10 minutes and then storage at 4°C.
Expression analysis
EC culture (Chapman et al.,
2001
), grafting
(Garcia-Martinez et al., 1993
),
and in situ hybridization (ISH) (Chapman et
al., 2001
; Chapman et al.,
2002
) were carried out as described previously.
Immunocytochemistry (ICC) was performed on whole-mount embryos or paraffin
sections (12 µm) using an anti-GFP antibody from Molecular Probes, at 1:400
dilution, with a secondary goat anti-rabbit Alexa Fluor® 488-conjugated
antibody (Molecular Probes), or with a HRP-conjugated goat anti-rabbit
secondary antibody for DAB labeling, at 1:200 dilution, according to our
standard protocol (Lopez-Sanchez et al.,
2004
). Briefly, for whole-mount immunocytochemistry, after
fixation in 4% paraformaldehyde (PFA) overnight, embryos were washed in PBS,
then washed for 4x30 minutes in PBT (PBS, 0.1% Tween-20 and 0.2% BSA),
and then 5% goat serum/PBT before addition of the primary antibody and
incubation overnight at 4°C. After washing for 4x30 minutes in PBT,
and once in 5% goat serum/PBT, the secondary antibody was added and embryos
incubated overnight at 4°C. For fluorescence visualization, embryos were
rinsed several times with PBS and imaged using a Nikon SMC1500 microscope,
with a GFP filter and a QImaging RTV5.0 Megapixel camera. A standard exposure
time of 20 seconds was used for fluorescence imaging to enable a comparison of
the fluorescent signal strength between embryos. For developing the DAB stain,
embryos were rinsed for 3x5 minutes in PBT, 4x30 minutes in PBT,
and for 10 minutes in 0.3 mg/ml DAB in PBT and then developed using 0.03%
hydrogen peroxide in DAB/PBT. Once the desired staining was reached the
reaction was stopped by several rinses in PBT and embryos were fixed in 4%
PFA/PBT before imaging and paraffin sectioning. For immunohistochemistry on
sections, slides were deparaffinized for 2x5 minutes in Xylene,
2x5 minutes in 100% EtOH, 5 minutes in 95% EtOH, and 5 minutes in 3%
H2O2/MeOH, then rinsed three times in distilled water,
followed by three times in PBS. Sections were then incubated at 37°C for 1
hour in 5% goat serum/PBT (PBS, 0.1% Triton and 0.2% BSA), and then 2 hours in
PBT with primary antibody diluted 1:400. After rinsing three times in PBT, the
sections were incubated with a fluorescent secondary antibody, diluted 1:200,
for 1 hour at 37°C, before washing for 3x5 minutes in PBS and
mounting in Slowfade (Molecular Probes) for visualization and imaging.
 |
Results
|
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An HIV-1 derived lentiviral vector (Tranzyme LLC; RTP, North Carolina) was
used because of its ability to integrate into the genome of dividing and
non-dividing cells, significantly increasing the host cell range. Enhanced
green fluorescent protein (eGFP) was inserted into the replication-deficient
vector under the control of a phosphoglycerol kinase promoter (PGK).
Retroviral suspension (2-5 µl) was microinjected into the subgerminal space
of each windowed egg containing a stage X embryo
(Eyal-Giladi and Kochav, 1976
).
Following injection, eggs were resealed with OpsiteTM surgical tape and
incubated to hatching. Of the 473 microinjected eggs, 19 G0 chicks hatched
(4%) and were grown to maturity. Of these 19, semen was collected from six
mature roosters and analysed for the presence of the transgene by PCR
(Fig. 1A). Although three of
the roosters were positive, only one rooster (rooster 6) passed the transgene
onto its offspring when mated to wild-type hens. Blood samples from 637
offspring from this rooster were analyzed by PCR, revealing 4 positives
(0.63%). Positives were further identified by the presence of fluorescent
lymphocytes. Of the resulting G1 birds produced, two roosters and a hen
survived to adulthood, and the roosters have been crossbred to wild-type hens.
The semen of one G1 rooster was analyzed by PCR and demonstrated template
quantity dependence (Fig. 1B).
Analysis of 170 eggs hatched from eggs sired by one of the heterozygous G1
roosters showed 68 G2 offspring positive for the transgene by PCR (40%).
Mature offspring from heterozygote individuals have been crossbred to produce
homozygous G3 individuals with two goals: (1) to eliminate the need for
individual testing before experiments are performed (i.e. all G4 offspring
will be positive/homozygote for the transgene); and (2) to obtain brighter
fluorescence (owing to the double dose of GFP present in homozygotes).

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Fig. 1. PCR transgene analysis. (A) Semen from six chimeric G0 roosters was
analyzed by PCR for the presence of the transgene. Three roosters were
positive for the transgene in the germline, but only rooster 6 was able to
pass the transgene onto his offspring. (B) Two mature offspring from rooster 6
had their semen tested for the presence of the transgene. Increasing template
quantity from one of these G1 heterozygous roosters showed the expected
quantity-dependent band increase. Neg, negative control; Pos, positive
control; MW, molecular weight marker.
|
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Recent reports of GFP transgenic chicks have not demonstrated ubiquitous
tissue expression (Harvey et al.,
2002a
; Harvey et al.,
2002b
; McGrew et al.,
2004
; Sang, 2004
).
In principle, the PGK promoter used in this study should result in ubiquitous
expression of GFP from the onset of development. To test the timing of the
onset, and the extent, of GFP expression, embryos were harvested from
unincubated eggs and every two hours following incubation until HH stage 12
(48 hours) (Hamburger and Hamilton,
1951
). In early embryos, expression was initially weak;
development to at least HH stage 9 was required to produce a strong enough
signal to be reliably detected using the fluorescence microscope (see
Table 1). In total, GFP
fluorescence could be detected only in 25% (12/48) of live embryos analyzed by
fluorescence microscopy, whereas the use of an anti-GFP antibody (which
enhances detection of the signal) revealed that 52% of the embryos produced
GFP protein at these early stages. Expression was ubiquitous, and included the
embryonic and extraembryonic tissues. By HH stage 13, expression could be
reliably detected in all transgenic embryos by GFP fluorescence. Thereafter,
the strength of the fluorescent signal continued to increase until
approximately HH stage 16/17 (Fig.
2A-D), after this stage the signal stabilized and remained at a
constant level during the remainder of development; E18 was the latest
developmental stage analyzed as whole embryos (E4-E18: 12/32 embryos were GFP
positive). Organs, including heart, lung, kidneys, liver, pancreas, intestine,
gizzard, eye and brain, were dissected from E14 embryos for more detailed
analysis. Fresh, unfixed organs from GFP-positive embryos were individually
analyzed and all were found to have uniform GFP expression in their organs by
fluorescence microscopy (Fig.
3A,B,D,E,G,H,J,K), whereas no organs were GFP-positive from
embryos that had no whole mount GFP-expression or from control non-transgenic
embryos (data not shown). Following processing of GFP-postitive organs by
paraffin histology, 12-µm sections were cut and subjected to
immunohistochemistry using an anti-GFP antibody. Secondary detection of the
antibody using an Alexa Fluor® 488-conjugated antibody and fluorescence
microscopy revealed GFP in all cells of the tissue sections. Dense cell
clusters appeared brighter than tissue areas with a low cell density. Sections
from control embryos had no fluorescence (data not shown). These results
demonstrate that the PGK promoter is effective at producing ubiquitous
expression in embryos from unincubated stages onward.
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Table 1. Numbers of living embryos with detectable GFP fluorescence and the same
embryos with enhanced fluorescence following processing using a rabbit
anti-GFP antibody and secondary anti-rabbit Alexa Fluor 488 antibody
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Fig. 2. GFP expression in whole-mount and sectioned transgenic embryos. (A-D)
Examination by brightfield (A,C) or fluorescence (B,D) illumination of a live
whole-mount 72-hour embryo (A,B) and a 72-hour embryo after sectioning and
processing with anti-GFP antibody (C,D). GFP expression is ubiquitous (B,D).
White line in A shows angle of the section. Scale bar: in A, 150 µm for
A,B; in C, 100 µm for C,D.
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Fig. 3. E14 chick organs express GFP uniformly. Fresh organs from E14 embryos were
dissected out and checked for GFP using fluorescence microscopy. Kidneys
(A-C), eye (D-F), heart (G-I) and brain (J-L) are all positive for GFP
expression. Lung, liver, pancreas, intestine and gizzard also uniformly
express GFP (not shown). (C) Transverse section of the left kidney shown in A.
(F) Magnified image of the right anterior portion of the transverse section of
the eye in D. The heart in G was sectioned through the plane of the paper. L
is an oblique section through the caudal part of the brain in J, including the
tectum, cerebellum and brain stem. Lines in A,D and J indicate level of
sections in C,F and L, respectively. a, apex; bs, brain stem; c, choroid; cb,
cerebellum; ch, cerebral hemisphere; l, lens; ms, mesonephros; mt,
metanephros; ot, outflow tract; r, retina; rpe, retinal pigment epithelium; s,
sclera; t, tectum; v, ventricle. Scale bars: in A, 150 µm for A,B,D,E,G-K;
in C, 50 µm; in F, 40 µm; in L, 35 µm.
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Fate mapping of embryos by transplanting cells carrying a stable tissue
marker is an obvious application of this technology. By transplanting cells
from chick donors to chick hosts potential confounding factors can be avoided,
such as potential species differences that might exist, for example, with
traditional quail/chick transplantation chimeras. Transplanted cells can
potentially be followed in the live embryo by fluorescence, processed for
molecular markers using RNA probes or antibodies, and also permanently marked
for identification following sectioning. Our interest in early development
lead to transplant experiments to determine the ability of GFP cells to
integrate into non-transgenic embryos. Cells were followed by fluorescence
microscopy and embryos were processed for in situ hybridization and
immunocytochemistry.
Transplant potential was tested using non-transgenic hosts and potential
transgenic donors placed into EC Culture
(Chapman et al., 2001
) at HH
stage 3. Hosts had a portion of the rostral primitive streak removed and
replaced with a homotopic and isochronic tissue graft from donor embryos
(Fig. 4A). Only 50% of donor
embryos were positive for GFP because a heterozygous male had sired the
transgenic embryos. Donor tissue was not scored for GFP, as the embryos were
insufficiently fluorescent for positive embryos to be reliably detected by
fluorescence microscopy alone (i.e. in the absence of anti-GFP antibody). In
14 grafted embryos, 12 had good integration of the graft
(Fig. 4B), and nine of these
were GFP positive after ICC processing using either a fluorescent secondary
antibody (n=3/4) or a DAB-labeled secondary antibody
(n=6/10) to produce a permanent marker
(Fig. 4C,D). Fluorescence or
DAB was seen in 11/22 potential donor transgenic embryos used as positive
controls, but not in embryos where the primary or secondary antibody had been
omitted (n=4). Another experiment used somite transplants excised
from GFP-positive 48-hour embryos. GFP-positive somite grafts were placed into
six wild-type host embryos into the caudal segmental plate mesoderm. Following
overnight incubation, the five embryos with intact grafts were processed for
DAB labeling. All five of these embryos were positive for GFP (data not
shown). In another experiment where grafts were taken from 48-hour embryos
(Fig. 5A) without scoring for
GFP and then placed homotopically and isochronically, three out of seven
embryos were positive for GFP after processing by ICC
(Fig. 5B-D).

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Fig. 4. Primitive streak graft using transgenic chick tissue. Wild-type chick host
embryo at HH stage 3d grafted with a segment of the primitive streak obtained
just caudal to Hensen's node from a transgenic chick donor embryo. (A) The
location of the grafted tissue in the host (white arrowheads) is shown
immediately after grafting. After 1 hour the graft had fully integrated. (B)
The grafted embryo after 20 hours of incubation in EC culture. (C) Following
immunocytochemistry with anti-GFP antibody and DAB, the grafted cells can be
identified within the heart tube and as bilateral streams extending caudally
into the area pellucida. The horizontal line indicates the level of the
section shown in D. (D) Paraffin sections of the embryo (12 µm) show that
the labeled cells (brown) contributed mainly to the heart mesoderm at the
level (anterior intestinal portal) of the fusing lateral body folds. Scale
bars: in A,D, 150 µm; in B, 250 µm for B,C.
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Fig. 5. Somite graft using transgenic chick tissue. (A) Three somites from a
transgenic embryo (black arrowheads) were grafted into the left side of a
48-hour wild-type chick host embryo (ventral side up in EC culture). (B) After
a 24-hour incubation, the integrated graft was observed on the left side of
the whole-mount embryo (dorsal view) after processing for immunocytochemistry
with an anti-GFP antibody and DAB staining. (C) Higher magnification view of
the integrated graft in B. (D) Section at the level indicated by the
horizontal line in B. Paraffin section (12 µm) at the level of the donor
somitic tissue demonstrates that cells integrate into the host embryo in the
neural tube, dermomyotome and heart. Scale bars: in B, 200 µm for A,B; in
C, 200 µm for C,D.
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Fate mapping relies on being able to process embryos after fixation and to
molecularly identify grafted cells by ISH.
Transgenic embryos were scored for GFP by fluorescence microscopy, fixed
and processed by ISH for Ganf (n=7), Sox2 (n=12), Dkk1
(n=7) and Chd (n=7), and then subjected to anti-GFP ICC
using Alexa Fluor® 488-tagged secondary antibodies
(Fig. 6A-F). The anti-GFP
antibody was required as fixation and ISH quench GFP fluorescence. In all
cases, the fluorescence was detectable following ISH and ICC, demonstrating
that tissue from transgenic embryos can be identified both by the GFP marker
using ICC, and molecularly by ISH.

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Fig. 6. In situ hybridization (ISH) and GFP expression in wholemount embryos. Chd
(A,B), Ganf (C,D) and Sox2 (E,F) were used as ISH probes in whole-mount
embryos. Anti-GFP antibody with an Alexa Fluor® 488-tagged secondary
antibody was used to restore fluorescence following ISH (B,D,F). Scale bar: in
F, 200 µm for A-F.
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Discussion
|
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We have produced GFP heterozygous transgenic chickens to the G2 generation,
and G3 homozygote birds that are due to mature and begin egg production soon,
thus demonstrating the feasibility of using lentiviral vectors to transduce
exogenous genes into the chick genome. Transgenic chickens are an ideal
bioreactor for the production of pharmaceutical products, and, together with,
a variety of permanent cell markers, will be a useful additional tool to
developmental biologists. The results of our culture, transplant, ISH and ICC
experiments demonstrate that the PGK promoter allows for ubiquitous expression
in embryonic tissue from unincubated stages up to E18. With the first draft of
the chicken genome now available, the potential for using lentiviral vectors
to produce chickens that can be used in analyzing gene function is timely.
Other markers that may benefit cell-tracing experiments using transgenic
chickens include, but are not limited to, ß-gal, DSRed, YFP, RFP and
other variants. Another useful addition would be the production of a Cre/Lox
chick, where microinjection of Tamoxifen, for example, would activate the
marker in a localized position. For example, in cells expressing Shh,
injection of tamoxifen would turn the marker on, and even if the cell later
turned off Shh expression the marker would remain, allowing identification of
cells.
Once licensing agreements are finalized, eggs from transgenic birds will be
available for distribution to interested parties
(www.geneworks.net).
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ACKNOWLEDGMENTS
|
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This work was funded by the NIH, grant numbers DC04185, DK065941 and
DK066445.
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