retrotrotransposable
elements (VL30’s) present at 100 to 200 copies in the genomes of most species
(Keshet et al, 1980; Courtney et al, 1982). Subsets of these endogenous
elements are naturally expressed in many tissues (Norton et al, 1988; Nilsson
and Bohm, 1994). During retroviral infections, mouse VL30 RNA is efficiently
transmitted by pseudotyping (co-packaging) into retrovirus particles (Howk et
al, 1978; Besmer et al, 1979; Scolnick et al, 1979). Vectors derived from
murine VL30’s are expressed in a variety of mammalian cell types, both primary
and transformed (Chakraborty et al, 1993, 1995).
These
characteristics of VL30 suggested that vectors derived from these elements
might give sustained expression in vivo. To test this hypothesis, a construct
expressing LacZ from a VL30 LTR was packaged using a standard retroviral
packaging cell line, and the resulting virions were used to transduce murine
embryonic stem cells. Selected transductants were used to derive a transgenic
animal via injection of ES cells into 3.5 day blastocysts and surgical
reimplantation. Sustained and tissue specific expression was observed in the
resulting transgenic mouse line. These results illustrate the utility of VL30
derived retro-vectors for obtaining transgene expression in vivo.
II. Results
A. derivation of vector and construction of the transgenic mouse line
The vector used
in these studies, which is referred to as VLSAIBAG (Figure 1A), was derived from the endogenous VL30 element NVL-3
(Carter et al, 1983). It contains a LacZ reporter gene expressed from the NVL-3
LTR. As VL30 RNA is poorly translated into protein (unpublished observations),
an internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV)
was cloned 5’ of the LacZ open reading frame to ensure efficient translation of
the reporter construct (Grunkemeyer et al, submitted). Embryonic stem cells
were transduced using viral supernatants from PA317/VLSAIBAG vector producer
cells (VPCs). A number of clonal cell lines were obtained by G418 drug
selection. One such clone was selected for micro-injection into mouse
blastocysts, resulting in the production of a single transgenic mouse line. The
transgene was integrated as a single copy, without rearrangements, in both the
ES cells (Figure 1B) and in the
resulting transgenic animals (Figure 1C).
Fluorescence in situ hybridization (FISH) analysis demonstrated that a single
autosomal integrant was stable through several mouse generations (Figure 2).
B. Analysis of vector expression in vivo
Preliminary
screening was carried out by reverse transcriptase polymerase chain reaction
(RT-PCR) on total RNA from various embryonic and adult tissues. These analyses
indicated expression in both spleen and lung (data not shown). RNase protection
analysis was then performed using total RNA isolated from lung and spleen
tissue from four and 15 week old animals. Figure
3A illustrates that a 194 bp protected fragment (corresponding to a portion
of the lacZ structural gene) was present in both lung and spleen tissue.
Significant amounts of vector RNA were observed in samples derived from both
young (29 day old) and mature (99 day old) mice, illustrating that expression
of the reporter gene is stable. To verify that the probe was detecting
full-length transcripts of the appropriate molecular size, RNA blot analysis
was performed using total RNA from lung tissue. Figure 3B demonstrates that the appropriate sized transcript (6.6
kb) was expressed in lung. Vector-derived transcript levels in the spleen were
too low to be detected by this method.
Expression of
vector-derived transcripts in RNA preparations from whole tissues might be
derived from a specific cellular compartment within the tissue. To delineate
transgene expression at the cellular level, we employed a histochemical
staining technique for enzymatically active b-gal protein. Results shown in Figure 4 demonstrated significant transgene expression in a subset
of cells in both lung (Figure 4B, and D)
and spleen (Figure 4F). Neither the
number of cells nor the intensity of staining appeared to vary significantly
between lung tissue derived from animals of two-weeks (Figure 4B) versus 10-weeks (Figure
4D) of age. No staining was observed in these same tissues from age-matched
non-transgenic littermates (Figure 4A, C
and E).
Based upon the
morphology, frequency, and localization of b-gal positive staining cells of the lung, we surmised
that they may be either macrophages or type II pneumocytes. To resolve this
issue, tissues were first stained with X-gal, followed by staining of sections
with antibodies specific for either macrophages or type II pneumocytes. The
same fields were observed by both light and fluorescence microscopy to
determine the identity of b-gal
positive cells. Analysis of Figure 5A,
and 5B clearly illustrates that
cells staining positive for the macrophage marker are not positive for b-galactosidase
activity. Similar analysis was carried out using an antibody specific for the
pro-peptide form of human surfactant protein C, which is an established marker
for type II pneumocytes (Vorbroker et al, 1995). Comparison of Figure 5C with Figure 5D illustrates that all blue cells were also positive for
surfactant protein C, identifying these b-gal positive cells as type II pneumocytes. It should be
noted, however, that not all antibody positive cells were blue, suggesting the
transgene was expressed in a sub-population of type II pneumocytes in the lung.
Similar
attempts to identify the Lac-Z positive cells in the spleen were inconclusive.
III.
Discussion
The results
described herein demonstrate that transgenic animals can be derived via
transduction of mouse embryonic stem cells using retro-vectors engineered to
express heterologous gene products from at least one VL30 promoter (NVL-3). In
the example described, a stably transduced ES cell clone was used to generate a
transgenic mouse which expressed theVL30 vector, VLSAIBAG, in a tissue-specific
manner. The retro-vector was integrated in single copy, and shown to pass
through the germ-line several times (>5) without affecting either the
expression levels or the cellular specificity of expression. Earlier attempts
to derive transgenic mice from embryonic cells transduced with retroviral
vectors resulted in inactivation of expression after passage through the germ
line (Jahner and Jaenisch, 1985). Inactivation was attributed to methylation of
both the retroviral genome and the DNA surrounding the site of integration,
based on direct studies as well as experiments where the retroviral genome was
activated by chemical demethylation of the DNA with 5-azacytidine (Jahner et
al, 1982; Jahner and Jaenisch, 1985). Thus, this method for producing
transgenic animals has been largely abandoned. VL30-derived vectors offer the
opportunity to revisit this strategy.
The
use of retroviral transduction of embryonic stem cells as a means of generating
transgenic animals has obvious advantages over methods currently employed. The
vector can be inserted in a single copy, avoiding complications due to
concatamerization of the transgene. It allows for testing of gene therapy
vectors in vivo, allowing quick
assessment of tissue specificity and toxicity. There are more than 100 copies
of VL30 in the mouse genome with wide ranging developmental and tissue specific
expression patterns (Sanes et al,
1986; Norton and Hogan, 1988; Nilsson and Bohm, 1994), and inducibility
(Rodland et al, 1986, 1988; Lenormand et al, 1992), suggesting that their use
in transgenics might have broad applicability.
In an earlier
report, Nilsson and Bohm, (1994) examined the endogenous expression patterns
for a number of the known VL30 elements in mouse tissues. They demonstrated
expression of the subclass of VL30 LTR’s that includes NVL-3 are expressed in
the lung and spleen. Our construct employed the NVL-3 LTR, and as reported
herein, was expressed in both the lung and the spleen. Nilsson and Bohm did not
resolve expression at the cellular level in these tissues. Our results suggest
that the NVL-3 LTR is regulated in the appropriate developmental and tissue specific
manner in the transgenic mouse line. However, because only one transgenic line
was analyzed, it is not possible to say with certainty that the transgene
functioned in a position-independent manner. Generation and analysis of more
transgenic lines, derived from indepentdently-transduced ES cell clones, are
necessary to resolve this issue. Indeed, expression of other retro-vectors have
been shown to be sensitive to position effects (Hoeben et al, 1991). However,
the fact that expression was observed in the same two tissues as observed
previously by Nilsson and Bohm, (1994) strongly suggests that the LTRs are
specific for these tissues.
This is the
first time that tissue-specific expression of a transgene has been demonstrated
using a VL30-derived vector. These findings point toward the use of
VL30-derived vectors for the generation of transgenic animals expressing a
heterologous gene in a tissue-specific or developmental stage-specific pattern.
The large number of endogenous VL30 LTRs which have been shown to be expressed
in distinct tissues at various times of development, may serve as a reservoir
of promoters for transgenic constructs as well as potentially being useful for
gene therapy.
Expression
of the retro-vector in type II pneumocytes illustrates highly specific cellular
expression of the NVL-3 LTR promoter. Indeed, only a subset of pro-SPC-positive
type II pneumocytes were positive for b-gal staining. Currently, pro-SPC is an accepted marker for
the identification of type II pneumocytes (Vorbroker et al, 1995). Our data
suggests that the capacity to express the retro-vector may delineate a subtype
of type II pneumocyte or simply that vector expression in some type II pneumocytes
is too low to be detected by the employed methods. Targeting expression to type
II pneumocytes might provide therapeutic angles for diseases such as pulmonary
emphysema, forms of which have been attributed to defects in matrix
metalloproteinase expression (Ohnishi et al, 1998).
IV. Materials
and Methods
A.
Vector construction
All plasmids were constructed according
to standard protocols (Ausubel et al, 1995). Plasmid pVLBAG was generated by
cloning a blunted lacZ/simian
virus-40 (SV40) early region transcriptional promoter/neomycin
phosphotransferase resistance cassette from pDOL (Price et al, 1987) into the unique NotI site of plasmid pVLPP (Chakrablrty
et al, 1993). Plasmid pVLPP is a
synthetic VL30 vector containing the LTRs and psi packaging signal from the murine retrotransposon, NVL-3 Carter
et al, 1983). Plasmid pVLIBAG was then constructed using pVLBAG as vector. A
blunted 601 bp NcoI-SalI fragment (from pG1IL2EN) (Treisman
et al, 1995), containing the IRES sequence from encephalomyocarditis virus
(Jang et al, 1988), was ligated into the unique PacI site in pVLBAG immediately 5’ of the b-gal initiator AUG codon.This clone was
used as vector for cloning pVLSAIBAG, the vector used in these studies. A 26 bp
splice acceptor fragment was cloned immediately 5’ of the IRES. The splice
acceptor was found to be non-functional both in vitro and in vivo (unpublished
observation). For RNase protection assays, plasmid pRIBOGAL was constructed by
cloning 194 bp of the lacZ coding
sequence into pBluescriptII (Stratagene, La Jolla, CA). All clones were
verified by restriction and sequence analysis.
B.
Viral transductions
The embryonic cell line RW4 (Genome
Systems, St. Louis, MO) was cultured according to the method of Robertson,
1987. ES cells were transduced by supernatants according to standard protocols
(Cepko et al, 1995). Viral supernatants were harvested from rapidly growing
(just confluent) cultures of PA317s, filtered through 0.2 m filter (Nalgene, Milwaukee, WI), and
added to ES cell cultures which had been passed onto gelatinized plates. The
following day, the cells were washed twice with PBS and allowed to grow for 48
hours. Transduced ES cells were selected in 175 mg/ml G418 for 10 days. G418 resistant
clones were expanded, and DNA (Puregene kit; Gentra Systems, Inc., Research
Triangle Park, NC) was isolated for determination of vector integrity (no major
rearrangements or deletions) and copy number. Clonal cell lines with correct
morphology and a single integrated copy of VLSAIBAG were expanded for injection
into pre-implantation embryos.
C.
Genetic typing
Hemizygote (designated +Z) males and
females were maintained as breeders in the colony so that each litter had a
likelihood of providing wild-type (++) and homozygous (ZZ) animals for experimentation.
DNA was isolated from tail DNA according to the following method: an
approximately 1 cm piece of tail was clipped with a sterile scalpel and
incubated in 0.5 ml digestion buffer (50mM Tris-Cl, pH 7.5; 50 mM EDTA; 1% SDS
and 10 mg/ml Proteinase K, Boehringer Mannheim) overnight at 37oC
and purified as described previously (Hogan et al, 1994). Tail DNA was digested
with HindIII, electrophoresed, and
hybridized to 32P-labelled probes for both the lacZ and neo coding
sequences. After exposure of autoradiographic film, blots were exposed to a
phospor screen to quantify the amount of signal in each lane and enable the
distinction of hemi- and homozygous animals.
D. Fluorescence in situ hybridization analysis
Cells were prepared for FISH and
G-banding (Figure 2C) analysis as
previously described (Takashi et al, 1991). Briefly, splenocytes were cultured
for 2-4 days in the presence of concanavalin A and lipopolysaccharide to
stimulate cell division. Metaphase spreads were prepared by synchronizing the
cells with 300 μg/ml thymidine, culturing in 5-bromo-deoxyuridine (BrdU)
and arresting them at metaphase with colchicine. The spreads were subjected to
FISH analysis as described.
The spreads were heat denatured and
hybridized to a digoxigenin-labelled probe (dig-dUTP; Boehringer Mannheim),
which was generated by nick translation of the lacZ probe (used for Southern and Northern analyses). After
hybridization, slides were washed and incubated with a fluorescein-conjugated
anti-digoxigenin antibody. The slides were viewed on an Olympus BH-2 microscope
equipped for epi-illumination. DAPI (4,6 Diamidino-2-phenylindole) was used as
a counterstain for the chromosomes (excitation = 367 nm, emission = 453 nm).
FITC (Fluoroscein isothiocyanate) was used to label the probe (excitation = 497
nm, emission = 524 nm). Low light level fluorochrome signals were captured and
enhanced using the Cytovision system (Applied Imaging Inc., Pittsburgh, PA).
E.
Northern blot and RNase protection analyses
Isolation of RNA from tissues was done
using TRIzoltm reagent (InVitrogen Corp, Gaithesburg, MD) according
to the manufacturers instructions. Total RNA was fractionated by
electrophoresis on agarose-formaldehyde gels (20 mg sample/lane). Blots were
UV-crosslinked, air-dried and hybridized to a probe for the lacZ coding sequence. RNase protection
assays were performed using the same total cellular RNA used for Northern
analyses with a kit (RPA II kit) from Ambion, Inc. (Austin, TX) according to
the manufacturer’s instructions. Antisense riboprobes were synthesized in vitro using T3 RNA polymerase
(Boehringer Mannheim), labeled a32P-UTP (Amersham), and linearized
plasmid pRIBOGAL. The riboprobe contains extra vector sequences, so undigested
probe (213 bases) can be differentiated from fully protected probe (194 bases)
on a denaturing acrylamide gel.
F. Histochemical stain
for b-gal activity and
visualization
Histochemical staining of tissues with
X-gal was performed as previously described (Sanes et al, 1986). Tissues were
fixed in 4% paraformaldehyde (PFA, Sigma Chemical Co.) for 1-2 hours at room
temperature. After fixation, tissues were rinsed four times in PBS and
incubated overnight at 30oC in X-gal stain solution [5.0 mM K3Fe(CN)6;
5.0 mM K4Fe(CN)6; 1.5 mM MgCl2; 0.02% NP-40;
0.01% sodium deoxycholate with 1.0 mg/ml X-gal (in DMSO) in PBS]. After
staining, tissues were rinsed in PBS and embedded in Historesin using the
methods described by the manufacturer (Leica; Heidelberg, Germany). Tissue
blocks were sectioned at 2.5 mm with a Sorvall JB-4 microtome (Ivan
Sorvall, Inc., Newton, CT) using a glass knife. To visualize cell morphology of
cells stained blue (due to b-gal activity) in the context of the
surrounding cells, each section was stained with Harris’ hematoxylin and
counterstained in alcoholic eosin according to standard protocols (Allen et al,
1992). Sections were coverslipped with an organic mounting medium (Curtin
Matheson Scientific, Inc., Houston, TX) and visualized on an Olympus BH2 oil
immersion microscope fitted with a digital camera and imaging system
(Cytovision, Applied Imaging, Pittsburgh, PA).
G.
Immunohistochemical staining and visualization
Tissues stained with X-gal were also
prepared for immunohistochemical analysis. After staining, tissues were rinsed
in PBS and embedded in Tissue-Tektm (Miles Inc., Elkhart, IN)
embedding medium. Tissues were frozen and stored at -150oC and
warmed to -25oC prior to sectioning. Blocks were sectioned at 3 m on a Microm HM 505 N cryostat (Carl
Zeiss, Inc., Thornwood, NY)
Various antibodies/antisera were used
to identify the specific cell types that express the vector VLSAIBAG in vivo. A polyclonal antisera against
mouse macrophages (a-mac; Accurate Chemical and Scientific Corp., Westbury, NY),
and a polyclonal antibody against the pro-peptide of human surfactant protein C
(a-hpro-SP-C, Vorbroker et al, 1995) were used for
immunohistochemical analysis on X-gal stained tissue sections. The basic method
is as described (Watkins, 1989).
For the a-mac antisera, normal goat
immunoglobulin G (Rabbit IgG, Vector Laboratories, Inc., Burlingame, CA) was
diluted to 12.5 mg/ml in 7% non-fat dry milk in PBS (milk/PBS) and incubated
on the section for 10-30 minutes at room temp. Next, the primary antibody
(polyclonal antibody/antisera) was diluted (1:300 dilution) in milk/PBS and
placed on each section. Sections were incubated with the primary antibody
overnight at 4oC. After incubation with the primary antibody, the
sections were washed three times in PBS before the FITC-conjugated (fluorescein
isothiocyanate) secondary antibody was applied to the sections. The secondary
antibody was diluted 1:100 in milk/PBS and incubated on each section for
2.5-3.5 hours at room temperature in the dark. Finally, the slides were washed
three times in PBS and coverslipped with Vectashieldtm (Vector
Laboratories, Inc., Burlingame, CA) mounting medium. The slides were documented
with an Olympus BH2 oil immersion microscope fitted with a digital camera and
imaging system (Cytovision, Applied Imaging, Pittsburgh, PA).
For the a-hpro-SP-C polyclonal, sections were
blocked (2% normal goat serum; 0.2% Triton X-100 in PBS) for 90 minutes at room
temperature before the addition of a-hpro-SP-C (dilution of 1:1000 in 2%
normal goat serum; 0.2% Triton X-100 in PBS). Primary antibody was incubated
overnight at 4oC. The next morning, the slides were washed with 0.2%
Triton X-100 in PBS before addition of the FITC-conjugated secondary diluted
1:100 in PBS containing 0.2% Triton X-100. After 2.5 hours at room temperature
in the dark, slides were washed with 0.2% Triton X-100 in PBS (1 wash for 5
minutes), PBS alone (2 washes for 5 minutes each) mounted with Vectashield,
coverslipped and imaged.
Acknowledgements
JAG was
supported by an NIH graduate fellowship through the BTNRH RTC P60 DC00982 from
the NIDCD. Work was supported by NIH R01 DK55000 and NIH P01 DC01813 to D.C.,
and by R29 GM41314 and a state of Nebraska Smoking and Tobacco-Related Disease
grant to C.P.H. We acknowledge the kind gifts from Dr. C. Cepco (pDOL) and
Jeffery Whitsett (anti-human pro-SP-C antibody). We are grateful to Jeffery
Pinnt for FISH analysis and to John (Skip) Kennedy for artwork.
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