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EMBO reports 4, 11, 1054–1058 (2003)
doi:10.1038/sj.embor.7400007
Published online: November 2003
Efficient transgenesis in farm animals by lentiviral vectors
Andreas Hofmann1, 2, 4, Barbara Kessler2, 4, Sonja Ewerling2, 3, Myriam Weppert2, Barbara Vogg1, Harald Ludwig1, Miodrag Stojkovic2, Marc Boelhauve2, Gottfried Brem3, Eckhard Wolf2 & Alexander Pfeifer1
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1 Department of Pharmacy, Institute for
Pharmacology, Center for Drug Research, Butenandtstrasse 5 (C),
Ludwig-Maximilians University, 81377 Munich,
Germany
2 Institute of Molecular Animal Breeding/Gene
Center, Feodor-Lynen-Strasse 25, Ludwig-Maximilians University,
81377 Munich, Germany
3 apoGene GmbH & Co. KG,
85354 Freising, Germany
4 These authors contributed equally to this
manuscript
To whom correspondence should be addressed
Eckhard Wolf Tel: +49 89 2180 76801; Fax: +49 89 2180 76849;
ewolf@lmb.uni-muenchen.de
Alexander Pfeifer Tel: +49 89 2180 77654; Fax: +49 89 2180 77326;
alexander.pfeifer@cup.uni-muenchen.de
Received 24 July 2003; Accepted 10 September 2003; Published online November 2003.
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Abstract
Microinjection of DNA is now the most widespread method for generating
transgenic animals, but transgenesis rates achieved this way in higher mammals
are extremely low. To address this longstanding problem, we used lentiviral
vectors carrying a ubiquitously active promoter (phosphoglycerate kinase,
LV-PGK) to deliver transgenes to porcine embryos. Of the 46 piglets born, 32
(70%) carried the transgene DNA and 30 (94%) of these pigs expressed the
transgene (green fluorescent protein, GFP). Direct fluorescence imaging and
immunohistochemistry showed that GFP was expressed in all tissues of LV-PGK
transgenic pigs, including germ cells. Importantly, the transgene was
transmitted through the germ-line. Tissue-specific transgene expression was
achieved by infecting porcine embryos with lentiviral vectors containing the
human keratin K14 promoter (LV-K14). LV-K14 transgenic animals expressed GFP
specifically in basal keratinocytes of the skin. Finally, infection of bovine
oocytes after and before in vitro fertilization with LV-PGK resulted in
transgene expression in 45% and 92% of the infected embryos, respectively.
EMBO reports 4, 11, 1054–1058 (2003)
doi:10.1038/sj.embor.7400007
Published online: November 2003
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Introduction
The introduction of foreign genes into early embryos is a powerful
tool for developmental studies and for the production of transgenic animals.
Pronuclear injection of DNA into zygotes (Gordon et
al., 1980) is now the most widespread technique used for
generating transgenic mice. However, this technique has had only limited
success in higher mammals because of inherent technical problems and low
efficacy. Therefore, the costs for the production of transgenic livestock by
pronuclear DNA microinjection are immense.
An alternative method is viral transgenesis—the use of
recombinant viruses to deliver genes into the embryo. Retroviral vectors based
on Moloney murine leukaemia virus transfer genes efficiently into murine,
porcine and bovine embryos (Cabot et al.,
2001; Chan et al., 1998;
Jaenisch, 1976); however, retroviruses are subject
to epigenetic modification, and retroviral expression is shut off during
embryogenesis (Jaenisch, 1976) or shortly after
birth (Chan et al., 1998).
Vectors derived from lentiviruses (for a review, see
Pfeifer & Verma, 2001), which belong to the
family of complex retroviruses, have been shown to transduce human embryonic
stem cells and pre-implantation embryos of mice and rats (Lois et al., 2002; Pfeifer et
al., 2002). However, attempts to generate transgenic monkeys by
lentiviral infection of pre-implantation embryos were not successful and
resulted in gene transfer only into extraembryonic tissues (Wolfgang et al., 2001), raising the question whether
lentiviral vectors can be used for transgenesis in higher mammals.
We used lentiviral vectors to achieve reproducibly high transgenesis
rates in swine and efficient gene transfer into bovine embryos and oocytes.
Results
Pig embryos were collected from gonadotropin-stimulated donor animals
after artificial insemination. Single-cell embryos were infected with high
titre (109–1010 infectious units per
millilitre) recombinant lentiviral vector pseudotyped with vesicular stomatitis
virus envelope glycoprotein G (Naldini et al.,
1996) by injection into the perivitelline space. The lentiviral
vector carries the green fluorescent protein (GFP) reporter transgene, a
central polypurine tract (cPPT) and the post-transcriptional regulatory element
of woodchuck hepatitis virus to increase transduction efficiency (Follenzi et al., 2000; Zufferey
et al., 1999). The lentiviral vector construct LV-PGK contains
the phosphoglycerate kinase promoter to achieve ubiquitous expression of the
reporter transgene (GFP; Fig. 1A).
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Figure 1
Southern blot analyses and in vivo imaging. (A) The
lentiviral vector carrying the phosphoglycerate kinase promoter (LV-PGK, top).
Arrow, self-inactivating mutation; eGFP, enhanced green fluorescent protein;
LTR, long terminal repeat; ppt, polypurine tract; W, woodchuck hepatitis
responsive element; wavy lines, pig genome. Southern blot of
BamHI-digested genomic DNA (bottom) isolated from skin samples of
piglets generated by subzonal injection of LV-PGK (pregnancies I–IV) and
one age-matched control animal (WT). (B) Southern blot analysis of DNA
extracted from animal #407. Spleen, brain, pancreas, muscle and lung carry
one viral integrant. (C–I) In vivo fluorescence imaging.
GFP expression was observed by direct epifluorescence in the skin and claws of
#511 (C, left), but not in the age-matched control animal (C,
right). Inset shows the bright-field image. Green fluorescence was also
observed in the eye (D, E), gingival tissue and tongue (G,
H) of the transgenic animal, but not in the eye (F) and snout
(I) of the control.
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After injection of LV-PGK, embryos were transferred endoscopically
into hormonally synchronized recipient females. Six pregnancies resulted in the
birth of 46 piglets (Table 1). No significant
differences were observed between the number of pregnancies derived from
transfers of virus-injected and control (buffer-injected) embryos (not
shown).
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Table 1
Transgenesis rate and green fluorescent protein expression in the
46 piglets derived from subzonal injection of LV-PGK (six pregnancies) and 16
piglets from subzonal injection of LV-K14 (two pregnancies)
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To analyse lentiviral integration and the number of proviral
integrants, Southern blot analysis (Fig. 1A) was
performed on genomic DNA digested with BamHI, which cuts only once in
the lentiviral vector. Thirty-two (70%) of the animals that developed from
subzonal injection of LV-PGK carried the provirus (Fig.
1A, and data not shown). In these animals, the number of proviruses
present in the genome ranged from 1 to 20 copies, and the mean copy number was
4.6  0.9. Integrated lentivirus was detected in organs derived from all
three primary germ layers (mesoderm, ectoderm and endoderm) and extraembryonic
tissues (Fig. 1B, and not shown). The number of transgene
copies were identical in all tissues analysed (Fig.
1B).
Transgene expression was assayed first in whole animals by in
vivo fluorescence imaging (Pfeifer et al.,
2001). Of the 46 animals born, 26 pigs expressed GFP at levels
detectable by direct fluorescence, whereas the age-matched control animals did
not exhibit green fluorescence (Fig. 1C). In the
GFP-positive animals, all tissues accessible to this non-invasive
technique—skin, claws, eye, tongue and oral mucosa (Fig.
1C–I)—exhibited green fluorescence. Analysis of internal
organs from animal #511 by direct fluorescence imaging showed that GFP was
expressed in all organs (Fig. 2A–D). Skin
(ectoderm), cerebellum (neuro-ectoderm), kidney (mesoderm) and pancreas
(endoderm) showed strong green fluorescence, whereas organs of an age-matched
control did not fluoresce (Fig. 2A–D).
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Figure 2
Analysis of green fluorescent protein expression by fluorescence
imaging and immunohistochemistry. (A–D) Fluorescence imaging of
skin (A), cerebellum (B), kidney (C) and pancreas
(D). All organs of transgenic animal #511 fluoresced (A and
C, left; B and D, bottom), whereas no significant
fluorescence was detectable in the control organs (A and C,
right; B and D, top). Insets, bright-field photographs.
(E–L) Histological analysis of green fluorescent protein (GFP)
expression by direct fluorescence and immunohistochemistry. (E,
I) Expression of GFP in the skin. Epidermal and dermal cells are GFP
positive. The inset shows a representative example of an epidermal ridge with
epidermal (white asterisk) and dermal cells being GFP positive. (F,
J) Analysis of GFP expression in the cerebellum. Red arrowheads indicate
Purkinje cells; white asterisk, granule cell layer; inset, border between
molecular and granule cell layer. (G, K) Cortical sections of
transgenic kidney express GFP in glomeruli (red arrowheads), proximal tubules
(white arrows) and distal tubules (yellow arrow). The inset shows a higher
magnification of the lower left glomerulus. (H, L) Detection of
GFP- and insulin-expressing cells in the pancreas by anti-GFP (brown) and
anti-insulin staining (blue). Red arrowheads, double-stained cells; inset,
islet of Langerhans. Shown are fluorescence images of organs (top),
epifluorescence (middle) and immunohistochemistry against GFP (bottom) of
consecutive sections. Nuclear stain (blue staining in E–H), DAPI;
scale bars, 50  m.
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At a cellular level, GFP expression was analysed by direct
fluorescence imaging of sections and by immunohistochemistry. We focused on
skin, cerebellum, kidney and pancreas. In skin sections the transgene was
expressed in all layers of the epidermis and dermis (Fig.
2E,I). In the cerebellum, GFP expression was strongest in the granular
layer, where numerous granule cells were GFP positive (Fig.
2F,J; asterisk). Expression of GFP was also observed in Purkinje cells
(Fig. 2J, arrowheads) and in the molecular layer (Fig. 2F,J). Cortical sections of the kidney contained
GFP-positive cells in glomeruli and distal tubules (Fig.
2G,K, arrowheads and yellow arrow). The highest level of GFP expression
was observed in the proximal tubules (Fig. 2K, white
arrows). Sections of the pancreas showed bright fluorescence (Fig. 2H). Immunofluorescence staining with polyclonal
antibodies (Abs) against insulin performed simultaneously with GFP staining
using monoclonal anti-GFP Abs showed expression of the lentivirally delivered
transgene in insulin-expressing pancreatic islets (Fig.
2L, arrowheads).
To study whether recombinant lentiviruses can be used to express
transgenes in specific porcine tissues, we constructed a lentiviral vector
(LV-K14) in which transgene expression is driven by a skin-specific promoter.
We chose the human keratin K14 gene promoter because it has been shown to be
strongly active in the dividing cells of the epidermal basal layer (basal
keratinocytes), hair follicles and oral epithelia. Most importantly, the K14
promoter has been used to establish skin-specific expression of various
transgenes in mice (Munz et al., 1999;
Vassar et al., 1989). As described above,
LV-K14 was injected into the perivitelline space of single-cell porcine
embryos. Two animals were born alive that carried the LV-K14 provirus (#519
and #526). Fluorescence imaging showed that GFP was expressed in the skin
and snout of these piglets (Fig. 3A), whereas all other
tissues were GFP negative—including cerebellum, pancreas and kidney
(Fig. 3B–D). Epifluorescent examination and
immunostaining of frozen skin sections showed that GFP expression was limited
to the basal layer of the epidermis and hair follicles (Fig.
3E,I). Transgene expression was not detectable in suprabasal epidermal
layers and in the dermis. Sections of the cerebellum (Fig.
3F,J), kidney (Fig. 3G,K) and pancreas (Fig. 3H,L) were GFP negative. However, Southern blot analyses
of animal #526 showed the presence of an identical number of LV-K14
proviruses in all organs including pancreas, kidney and skin (Fig. 4A). Western blotting confirmed the restriction of
transgene expression to the skin in LV-K14 transgenic pigs (Fig.
4B).
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Figure 3
Tissue-specific transgene expression in LV-K14 transgenic pig.
(A–D) Fluorescence imaging of isolated tissues. The skin
(A, left) of pig #526 showed green fluorescence, whereas cerebellum
(B, left), kidney (C, left) and pancreas (D, left) did not
fluoresce. No fluorescence was detectable in the organs of the age-matched
control (A–D, right). Insets, bright-field photographs of the
corresponding fluorescence images. (E–L) Histological analysis of
green fluorescent protein (GFP) expression in animal #526. (E,
I) Analysis of transgene expression in skin sections. GFP expression was
only detected in the basal layer of the epidermis and hair follicles (I,
arrows). The inset shows a higher magnification of a typical junction of
epidermis (white asterisk) and dermis; the inset arrow indicates basal
keratinocytes. In the cerebellum (F, J), (asterisk, granule cell
layer), kidney (G, K) and the pancreas (H, L),
neither green fluorescence nor GFP-specific immunostaining were detectable.
Shown are fluorescence images of organs (top), epifluorescence images (middle)
and immunohistochemistry against GFP (bottom) of consecutive sections. Nuclear
stain (blue in E–H), DAPI; scale bars, 50 m.
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Figure 4
Analyses of transgene expression. (A) Southern blot analysis
of vector integration in pancreas, kidney and skin of animal #526 (see also
Fig. 3). (B) Western blot analyses of transgene
expression using anti-green flourescent protein (GFP) antibodies on tissue
samples isolated from a LV-K14 (#526, top) and a LV-PGK transgenic pig
(#511, bottom). (C) Correlation between proviral copy number and GFP
expression levels quantified by immunoblotting.
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To determine the effect of copy number on transgene expression levels,
we performed western blot analyses on tissue samples isolated from pigs
carrying LV-PGK (pregnancies I–IV, Fig. 4C).
Interestingly, the relationship between copy number and GFP concentration was
almost identical in low (for example, two integrants) and high (for example, 12
integrants) copy animals. Transgene expression was detected by immunoblotting
in 30 (94%) out of 32 transgenic animals and increased over a broad range
almost linearly with increasing lentiviral vector number (Fig.
4C, and data not shown). All animals that carried more than two proviral
copies expressed GFP (Fig. 4C and Table
1).
An important question is whether lentiviral gene transfer into
pre-implantation embryos results in germ-line transgenic animals. Direct
fluorescence imaging of sections of the testis of newborn pigs derived from
subzonal injection of LV-PGK revealed GFP-positive cells in the seminiferous
tubules (Fig. 5A,B). Staining with the Dolichos
biflorus agglutinin (DBA), which specifically binds spermatogonial cells
during the first weeks after birth (Ertl et al.,
1992), identified the GFP-positive cells as spermatogonia (Fig. 5C–F). To address whether the lentivirally delivered
transgene can be transmitted through the germ line, we obtained sperm from pig
#507 (see also Fig. 1A). The presence of LV-PGK in
spermatozoa was detected by fluorescence in situ hybridization (FISH)
analysis (Fig. 5G, inset). Flow cytometer analysis showed
that GFP was expressed in sperm of animal #507 (Fig.
5G). In addition, the transgenic sperm was used to inseminate one
wild-type female pig. Subsequently, embryos at the 2–4-cell stage were
isolated and analysed by nested PCR. We detected the transgene in 4 out of 11
embryos, which clearly shows that LV-PGK is transmitted through the germ line
(Fig. 5H).
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Figure 5
Analyses of transgene expression, transduction of germ cells and
lentiviral transduction of bovine blastocysts. (A, B)
Histological analysis of testicular sections of a LV-PGK transgenic piglet.
Green fluorescent protein (GFP)-positive cells can be seen in the seminiferous
tubules. Shown are the haematoxylin and eosin staining (A) and
epifluorescent image (B). Scale bar, 50 m. (C–F)
Detection of transgene expression in spermatogonia of a newborn LV-PGK piglet
(C, D), as detected by Dolchios biflorus agglutinin (DBA)
staining (red). Control spermatogonia are GFP-negative (E, F).
Scale bar, 50 m. (G) Flow cytometer analysis of GFP expression in
spermatozoa of a control animal and animal #507 (LV-PGK). Inset,
photomicrograph showing representative fluorescence in situ
hybridization results. Sperm nuclei of #507 bearing red signals for LV-PGK
and blue signals for the Y chromosome. (H) Detection of the transgene by
nested PCR in 2–4-cell stage embryos derived from a mating of animal
#507 with a wild-type female. Four of the 11 embryos carry the transgene;
+, positive control (skin sample of #407); 0, empty lane; 1–11,
embryos analysed. (I–L) Analysis of GFP expression in bovine
blastocysts derived from subzonal injection of zygotes (I, J) and
oocytes (K, L) with LV-PGK. Arrowheads and asterisk, GFP-positive
and -negative zygote-derived blastocysts, respectively. Shown are the bright
fields (I, K) and the fluorescence images (J, L).
Nuclear stain (blue in B and inset G), DAPI. Scale bar, 200
m.
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To determine whether lentiviral transgenesis could also work in other
relevant livestock species, we infected bovine zygotes or oocytes with the
lentiviral vectors. Two hundred and twenty-seven zygotes were injected
subzonally with LV-PGK and 45% 22% (n = 4 experiments) of the
in vitro developing blastocysts expressed GFP (Fig.
5I,J). Lentiviral infection of zygotes resulted in GFP-positive
blastocysts at more than double the efficiency of conventional pronuclear
injection (17%; Chan et al., 1998). The data
shown so far resulted from injection of virus after fertilization of oocytes.
Next, we injected lentivirus before in vitro fertilization, to allow for
viral integration into the bovine genome before a nuclear membrane encloses the
chromatin. Subzonal injection of LV-PGK into bovine oocytes resulted in the
expression of GFP in 92% 8% (n = 3 experiments) of the
developing blastocysts (Fig. 5K,L). Analysis of GFP
expression in blastocysts derived from lentivirus-injected oocytes and zygotes
by confocal microscopy clearly showed that GFP was expressed in the
trophectoderm as well as the inner cell mass (see
supplementary information
online). The percentage of embryos developing to blastocysts after virus
injection (40% 17%, n = 227) was not significantly different
when compared with control treatment (47% 16%, n = 83). So,
subzonal injection of lentiviruses does not affect embryonic development.
Discussion
The GFP transgene was not found in neonates developed from rhesus
monkey embryos infected with lentiviral vectors (Wolfgang
et al., 2001). Further analyses revealed that the transgene
was present only in extraembryonic tissues such as the placenta (Wolfgang et al., 2001). By contrast, we observed
efficient transduction of the embryo proper and extraembryonic cells after
infection of bovine embryos and oocytes. In transgenic pigs, the derivatives of
the three primary germ layers, germ cells and extraembryonic tissues contained
the provirus and expressed GFP. In addition, the lentiviral provirus was
transmitted through the germ line. The different efficacies of lentiviral
transgenesis in monkey versus livestock might be due to basic biological
differences as well as technical aspects such as the time-point of virus
injection (single-cell embryos versus blastocysts; Wolfgang
et al., 2001) and virus titre.
An important feature of lentiviral transgenesis in livestock is the
linear correlation of transgene expression and number of integrants. This shows
that lentiviral transgene expression is controlled predominantly by the number
of proviral integrants. In contrast to previous studies that used retroviral
vectors for generating transgenic livestock (Chan et
al., 1998), silencing does not seem to have an important role in
lentiviral transgenesis. In addition, ubiquitous as well as tissue-specific
expression can be easily achieved by incorporating different internal promoters
in the lentiviral vector construct.
Owing to the fact that transgenesis rates are the most important
determinant of production costs, the inefficiencies of pronuclear DNA
microinjection (only  1% to 10% of the injected embryos are transgenic)
mean that the production costs of transgenic pigs and cattle are very high
(Moffat, 1998; Wells et
al., 1999). Lentiviral transfer of genes into porcine and bovine
genomes resulted in a reproducibly high efficiency of transgenesis. On the
basis of previous estimations of the impact of transgenesis rates on production
costs, lentiviral transgenesis could reduce costs of transgenic livestock to
about one-tenth of the present costs (Wells et al.,
1999). Low production costs are essential for the widespread use of
transgenic swine as disease models and donor animals for xenotransplantation,
as well as for the genetic improvement of agricultural populations.
Methods
Virus production.
The vector LV-PGK (Follenzi et al.,
2000) was recently described. LV-K14 was constructed by replacing the
PGK promoter of LV-PGK with the promoter of the human K14 gene (Munz et al., 1999). Recombinant lentivirus was
produced as previously described (Pfeifer et al.,
2002).
Pig embryo collection and virus injection.
Embryos were collected from 6-month-old gilts after slaughter. These
donors were superovulated with 1200 IU gonadotropin (pregnant mare serum
gonadotropin, PMSG; Intergonan, Intervet, Unterschleissheim, Germany), and
ovulation was stimulated with 750 IE human chorionic gonadotropin (HCG;
Ovogest, Intervet) 3 days later. During the following 24–36 h donor
animals were artificially inseminated twice and slaughtered 36 h after the
first insemination. Their oviducts were flushed with 38 °C flush-media (PBS
supplemented with 20% lamb serum, Invitrogen, Karlsruhe, Germany) and 50 mg
gentamicin sulphate (Sigma, Germany). Embryos were collected in flush-media and
used directly for subzonal virus injection with glass capillaries containing
concentrated virus.
Pig embryo transfer.
Six-month-old prepuberal gilts were used as recipients and
synchronized by oral administration of altrenogest (Regumate, Serumwerk,
Bernburg, Germany) over a 15-day period, followed by administration of 750 IE
PMSG 1 day after the last gestagen feeding. Ovulation was induced 3 days later
with 750 IE HCG. Transfers were performed by laparoscopy under general
anaesthesia with a combination of 1.2 ml per 10 kg ketamine hydrochloride
(Ursotamin, Serumwerk) and 0.5 ml per 10 kg xylazine (Xylazin 2%, WDT, Germany)
injected intravenously. To each recipient, 30–40 injected or control
embryos were transferred in one oviduct.
In vitro production of bovine embryos.
Bovine cumulus oocyte complexes (COCs) were collected by aspirating
ovarian follicles obtained from slaughtered animals. The in vitro
production of bovine embryos was performed as previously described (Stojkovic et al., 2001). Briefly, oocytes were
matured in vitro for 22 h in modified tissue culture medium 199
(Invitrogen) at 39 °C in 5 % CO2.
After maturation, COCs were distributed among different treatment
groups. In group I, COCs were co-cultured with frozen–thawed semen
(106 spermatozoa per ml; capacitated in a swim-up procedure) for
18 h. Presumptive zygotes were then denuded by vortexing and injected
subzonally with LV-PGK. In group II, oocytes were stripped free from cumulus
cells and injected subzonally with LV-PGK; this was followed by in vitro
fertilization as described above. Embryos were cultured in modified synthetic
oviduct fluid supplemented with 10% (v/v) oestrous cow serum (ECS;
Stojkovic et al., 2001) at 39 °C in a
humidified atmosphere of 5% CO2, 5% O2 and 90%
N2.
Southern blotting and nested PCR.
BamHI-digested DNA was separated by electrophoresis and
transferred to Gene Screen Plus Hybridization Transfer Membranes (PerkinElmer,
Boston, Massachusetts, USA). The blot was hybridized with a full-length
32P-labelled enhanced GFP (eGFP) cDNA probe. For nested PCR,
porcine embryos at the 2–4-cell stage were isolated and incubated with
Protease K (Roche). Thirty cycles were performed with GFP-specific primers
(forward, 19–38 nucleotide (nt); reverse, 716–734 nt of the EGFP
cDNA); thereafter, 35 cycles were done using nested primers (forward,
150–168 nt; reverse, 542–561 nt).
In vivo fluorescence imaging.
Excitation of green fluorescence was achieved using a Schott 2500
light source and a 485-nm filter (Zeiss, Jena, Germany). The emitted
fluorescence was visualized using a long-pass filter (HQ 500, Zeiss).
Immunohistochemistry and FISH technology.
Frozen 10-m sections were incubated with antibody against eGFP
(Clontech, Palo Alto, California, USA), followed by incubation with the
secondary biotinylated antibody (goat anti-mouse, Dianova, Hamburg, Germany)
and staining with VECTASTAIN ABC Peroxidase Kit (Vector, Burlingame, CA, USA)
and 3',3-diaminobenzidine (270 g ml-1; Sigma).
Staining of pancreatic islets was performed using a polyclonal guinea pig
anti-insulin antibody (BioTrend, Köln, Germany). Direct fluorescence
imaging was performed on untreated sections after washing with PBS and mounting
(Permafluor, Shandon, Lipshaw, Pittsburgh, USA). Nuclear staining was carried
out with 1 g ml-1 4',6-diamidino-2-phenylindole
(DAPI).
For fluorescence in situ hybridization technology, sperm
samples were prepared as recently described (Pellestor et
al., 1996). A Y-chromosome-specific probe (Rubes et al., 1999) and a GFP-specific probe
(forward, 19–39 nt; reverse, 542–561 nt) were directly labelled
with either fluorescein-12-dUTP or tetramethyl-rhodamine-dUTP (Roche) in a PCR
reaction.
Western blotting.
Tissue samples were homogenized in lysis buffer (0.5% Triton X-100,
150 mM NaCl, 2 mM CaCl2 and protease inhibitors). After separation
on SDS–polyacrylamide gel electrophoresis (PAGE) and transfer to
polyvinylidene fluoride membranes (Immobilon-P, Millipore, Bedford, USA.), GFP
was visualized using eGFP antibodies (Clontech) and enhanced chemoluminescent
labelling (Amersham). Quantification of GFP expression was performed by
immunoblotting. Protein samples were diluted according to the number of
lentiviral integrants present in their genome, and limiting dilutions were
performed to ensure that the signal was in the linear range. GFP concentration
was calculated by comparing individual bands with the band intensity of a
recombinant eGFP standard (Clontech) and given as ng
g-1 total protein.
Supplementary
information is available at EMBO reports online
(http://www.emboreports.org).
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Acknowledgements
We thank H. Sebald and T. Holy for expert technical assistance. We
also thank the Bayerische Forschungsstiftung (BFS 219/96, 492/02), Fonds der
Chemischen Industrie and the Deutsche Forschungsgemeinschaft.
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