BIOLOGY OF REPRODUCTION 56, 119-124 (1997)
Adenovirus-Mediated Gene Transfer by Perivitelline Microinjection of Mouse,
Rat, and Cow Embryos'
H. Michael Kubisch,3 Melissa A. Larson, 3 Peggy A. Eichen, 3 James M. Wilson, 5 and R. Michael Roberts 2,3 4,
Departments of Animal Sciences3 and Biochemistry,4 University of Missouri-Columbia, Columbia, Missouri 65211
Institute for Human Gene Therapy and Departments of Medicine and Molecular and Cellular Engineering, 5
University of Pennsylvania Medical Center, and the Wistar Institute, Philadelphia, Pennsylvania 19041
ABSTRACT
To determine the fate of an episomally expressed transgene,
mouse, rat, and cow zygotes were injected into the perivitelline
space with approximately 100 p of buffer containing the replication-defective human adenovirus, AdCMVLacZ/sub360. Viral concentrations ranged from 2.5 to 2.5 x 105 plaque-forming
units (pfu)/100 pl. As viral titer increased, fewer embryos were
able to develop to blastocysts. In the mouse, the percentage of
blastocysts formed ranged from 82% in controls to 16% after
injection at the highest titer. In the rat and cow, a similar decrease in blastocyst formation was noted (62% to 6% and 26%
to 4%, respectively). Reporter gene (galactosidase, LacZ) activity could be detected in mouse embryos after injection at a concentration of only 25 pfu/100 pl, whereas a tenfold higher titer
was required in the other two species to observe the blue LacZ
reaction product. When examined after 5 (mouse), 6 (rat), or 9
(cow) days of in vitro culture, the proportion of LacZ-positive
embryos ranged from 15% to 96%, 6% to 76%, and 18% to
58% in mouse, rat, and cow embryos, respectively, depending
upon viral concentration. However, a large percentage of positive embryos proved to be expression mosaics, the degree of
which was likewise dependent on titer. While none of the embryos showed LacZ activity at 30 h after injection, 70% of
mouse, 8% of rat, and 20% of cow embryos expressed the reporter gene at 42 h. Delaying the timing of injection revealed
that the efficiency with which mouse and rat embryos could be
infected decreased with increasing degree of differentiation.
Only 35% and 18% of mouse embryos expressed the reporter
gene after injection at the morula or blastocyst stage, respectively. A similar drop in efficiency was noted in rat embryos
when injections took place at the 8-cell, morula, or blastocyst
stage, with 70%, 33%, and 9% of embryos, respectively, subsequently showing LacZ activity. Likewise, advanced development resulted in a decrease in the efficiency of viral-mediated
gene transfer in cow embryos, with 100%, 78%, and 68% of
embryos being positive after injection at the 8-cell, morula, or
blastocyst stage, respectively. These results demonstrate that a
human adenovirus can be used to express a reporter gene transiently in nonhuman embryos.
INTRODUCTION
There exists considerable interest in developing new
means of delivering transgenes either for studies on transient gene expression or for transformation of cells or
whole organisms. Gene transfer into embryos has relied on
three strategies. The most common approach is pronuclear
microinjection of DNA [1], while others have used retro-
viruses as vectors [2-6], or episomally maintained viruses,
such as the bovine papilloma virus [7]. While the efficiency
of introducing transgenes by microinjection, particularly
into farm animal species, is low when assessed as the number of transgenic animals born, it has been reported that
expression of constructs injected into the pronucleus can be
found in over 70% of mice and 20% of cow and pig embryos, with the majority of these being expression mosaics
[8-11]. These observations suggest three possible explanations: 1) that the transgenes become integrated after the
first cell division and hence are segregated into different
lineages; 2) that the putative transgenes are silenced in individual blastomeres as development progresses; 3) the
most likely possibility-that expression in these embryos
stems predominantly from nonintegrated DNA. The last
possibility would require that nonintegrated DNA be maintained through successive cell divisions and become unequally partitioned between blastomeres so that a decreasing population of cells maintains the episomic DNA as development proceeds.
To investigate the expression patterns of nonintegrating
exogenous DNA during subsequent cell divisions, we have
used the replication-defective recombinant adenovirus
AdCMVLacZ/sub360 to infect mouse, rat, and cow embryos by perivitelline injection. This serotype 5 virus has been
used to transfer genes into a variety of cells in different
species [12-16]. The process of internalization of the virus
is as yet poorly understood but appears to involve binding
to an unidentified primary receptor and subsequent endocytosis. The process may require expression of av35 integrin [17]. After internalization, adenoviral genomes do not
integrate into host chromosomes but are maintained episomally [18]. Thus, the use of episomally maintained viral
DNA may give insight into which of the three proposed
theories concerning mosaic expression of injected transgenes is correct.
In the AdCMVLacZ/sub360 virus, the El gene has been
replaced with the bacterial -galactosidase gene (LacZ)
linked to the human cytomegalovirus promoter. The product of the LacZ gene can be readily detected in embryos,
thus facilitating confirmation of the reporter gene [12].
The objectives of this study were to evaluate the ability
of AdCMVLacZ/sub360 to infect mouse, rat, and cow embryos at different developmental stages; to assess the effects of viral infection on subsequent development; and
lastly, to examine the patterns of reporter gene expression
at different viral titers.
Accepted August 23, 1996.
Received May 20, 1996.
'This project was supported by NIH grant HD21896 and a grant from
Genzyme Transgenics Inc. Contribution from the Missouri Agricultural
Experiment Station, Journal Series Number 12,485.
2Correspondence: Dept. of Animal Sciences, University of Missouri,
158 Animal Science Research Center, Columbia, MO 65211. FAX: (573)
882-6827; e-mail: [email protected]
MATERIALS AND METHODS
Generation and Culture of Murine Embryos
Female mice (NIH Swiss; Harlan Sprague Dawley, Indianapolis, IN) at 28 days of age were superovulated with
10 IU eCG followed by 5 IU hCG 48 h later. After being
119
120
KUBISCH ET AL.
left with males overnight, females were killed, and -cell
embryos were collected from the oviducts in CZB medium
[19] buffered with HEPES (Sigma, St. Louis, MO). These
were cultured for 5 days in CZB. If injection took place at
the blastocyst stage, culture was extended by an additional
day.
Table 1. Development and reporter gene expression in mouse, rat, and
cow embryos.
Embryo
A) Mouse
Generation and Culture of Rat Embryos
Female Sprague Dawley rats were obtained from a local
colony and were superovulated at 42 days of age by peritoneal injection of 25 IU of eCG followed by 25 U of hCG
50 h later. Matings were confirmed by detection of mating
plugs. Embryos were retrieved from oviducts in HEPESbuffered R1ECM medium and cultured for 6 days in
RIECM [20].
Generation and Culture of Bovine Embryos
Bovine ovaries were collected at an abattoir and transported to the laboratory at 30 0C in PBS (1.4 mM KH 2PO 4,
8 mM Na2 HPO 4, 0.14 M NaCl, 1.7 mM KCI). Ovarian
follicles measuring between 2 and 8 mm in diameter were
aspirated under a negative pressure of 2.5 psi. The follicular
fluid was pipetted through a 100-1Lm cell strainer (Becton
Dickinson Labware, Franklin Lakes, NY), and the cumulus
complexes were washed out of the strainer with Tyrode's
albumin lactate pyruvate (TALP)-HEPES [21]. Oocytes
were matured and fertilized as described previously [22].
Embryos were cultured for the first 48 h at 39 0 C in 5%
CO 2 in glucose-free CZB medium [19] that had been conditioned by Buffalo rat liver cells (BRL 3A, referred to
hereafter as BRL) for 36 h [22]. After 48 h in CZB, embryos were examined to assess development, and 8-cell embryos were selected and placed in TCM 199 medium (Gibco, Gaithersburg, MD) containing 10% fetal bovine serum
(Sigma) and 0.25 mM sodium pyruvate. This medium, too,
had been conditioned by BRL cells. Embryos remained in
culture for an additional 7 days.
Injection of Virus and Determination of Reporter
Gene Activity
The construction and propagation of the virus AdCMVLacZ/sub 360 are described elsewhere [12]. For injection, the virus was diluted from the stock concentration
of 2.5
1012 plaque-forming units (pfu)/ml with 0.9% saline to the desired concentration. Bovine embryos were
handled in TALP-HEPES. Handling medium for mouse
embryos was HEPES-buffered CZB, and rat embryos were
manipulated in HEPES-buffered R1ECM. All embryos
were microinjected while maintained in droplets of their
respective handling medium under paraffin oil. Bovine embryos were vortexed to remove cumulus cells and centrifuged at 13 000
g for 7 min to displace cytoplasmic
lipids and visualize both pronuclei to assure that fertilization had occurred. All injections were performed at 200
magnification under an inverted microscope (Nikon Instruments, Garden City, NY) with Hoffman Modulation Contrast optics. Injections were deemed successful when swelling of the zona pellucida could be observed. Although it
was impossible to assess exactly what volume of solution
was injected or how much was retained under the zona after
needle withdrawal, the degree of swelling suggested an increase in volume of approximately 100 pl. After injection,
embryos were returned to their respective culture medium
until they were scored and stained for reporter gene ex-
B) Rat
C) Cow
Titer
(pfu/100 pi)
Blastocysts
n (%)
Controls*
2.5
25
2.5 x 102
2.5 x 101
2.5 x 104
2.5 X 10)
41
42
29
31
28
19
8
Controls*
2.5
25
2.5 x 102
2.5 X 10 j
2.5 x 104
2.5 x 101
31 (62)'
23 (46)'
30 (60)a '1
Controls*
2.5
25
2.5 x 102
2.5 x 103
2.5 x 104
2.5 X 10'
13
10
8
7
(82)P
(84)"
(58)"
(62)1
(56)"
(38)"
(16)'
b
15 (30 )
13 (26)'
13 (26)'
3 (6)d
(26)"
(20),'1
(16) "b'
(14), b ,
b
5 (10) '"
c
3 (6) "
2 (4)o' d
LacZ positive
n (%)
0
0
8 (15) .~
43 (86)"'
37 (74) b
42 (84)"
48 (96)'
Mosaic
n (%)
7
22
20
5
8
(88)'
(51)
(54)'
(12)"
(17)"
0
0
0
3 (6)'
13 (26)"'
34 (68)'
38 (76)'
3 (100)'
5(39)"'
14 (41)1
3(8)'
0
0
0
9 (18)'
15 (30)"a
20 (4 0 )b'
29 (58)
8
12
8
14
(89)'
(80)'"
(40)'
(48)"
*Noninjected. Fifty zygotes were used for each viral titer examined. Embryos received perivitelline injections of approximately 100 p at the
1-cell stage.
.,,c.d Values with different superscripts in the same column and species
are significantly different at p < 0.05.
pression. At that time, embryos were fixed in 0.2% (w:v)
glutaraldehyde, washed twice in PBS, and stained in 0.1 M
phosphate buffer (pH 7.3) containing 1 mg/ml of 5-bromo4-chloro-3-indolyl-3-D-galactopyranoside (X-Gal; Sigma)
at 37°C in air for 12 h [23].
Data on development and expression were analyzed by
a chi-square procedure [24, 25].
RESULTS
Effect of Varying Titers on Development, and Degree and
Pattern of Reporter Gene Expression of
AdCM VLacZ/sub3 60
To determine the effects of viral infection on development and to determine the optimal titer for subsequent experiments, embryos received injections of viral titers ranging from 2.5 to 2.5
105 pfu/100 pl.
Mouse embryos. Groups of 50 zygotes were employed
for each titer of virus tested. The viral concentration had a
significant effect on the number of resulting blastocysts (p
< 0.01, Table 1A). The percentage of injected embryos that
developed to blastocysts in 5 days declined from 82% in
controls to 16% in the group that received the highest titer.
Viral concentration similarly affected the number of embryos that stained positively for the reporter gene. The expression of the LacZ gene was not detectable at a viral
concentration of less than 25 pfu/100 pl. LacZ activity increased significantly when injections were performed at
concentrations greater than 2.5 x 105 pfu/100 pl (p < 0.05,
Table 1A), but there was an accompanying loss in developmental potential. There was also an inverse relationship
between amount of virus injected and the degree of mosaicism of the embryos in terms of their LacZ expression.
Thus, mosaicism declined as viral titer was increased (Table
1A, Fig. 1A).
Rat embryos. Rat embryos received injections as above
but were stained at Day 6 of culture. As with mouse em-
ADENOVIRUS-MEDIATED GENE TRANSFER
121
FIG. 1. Murine, rat, and bovine embryos stained for LacZ activity following an earlier injection with AdCMVLacZ/sub360. A) Murine morulae after
injection at the zygote stage of a viral concentration of 2.5 x 104 pfu/100 pi (x75). B) Rat blastocysts after injection at the zygote stage of 2.5 x 104
pfu/100 pi (x100). C) Bovine embryos after injection at the zygote stage of 2.5 x 105 pfu/100 pi (XI 70). D) Bovine blastocysts after injection at the
blastocyst stage of 2.5 x 104 pfu/100 pi (X75).
bryos, viral titer determined how many of the embryos advanced to the blastocyst stage. There was a significant loss
of developmental potential with viral concentrations of 2.5
x 103 and 105 pfu/100 pl (Table 1B). A tenfold higher viral
titer (250 pfu/100 pl) was required to provide detectable
LacZ activity in rat embryos than in mouse embryos.
Again, however, there was an increase in the number of
embryos expressing LacZ and a decline in mosaicism as
viral titer was increased (Table 1B, Fig. 1B).
Bovine embryos. In these experiments, only 8-cell embryos were selected at 48 h for continued culture to the
blastocyst stage since previous experiments have established that embryos with fewer cells do not advance successfully. However, the data on LacZ expression in Table
1C represent all embryos, including those that were arrested
before the 8-cell stage. Viral titer again proved to affect
development to the blastocyst stage, which decreased to 4%
after injection at a titer of 2.5 X 105 pfu/100 pl. This value
contrasts with controls and with embryos that received the
lowest titer, in which between 20% and 26% of 1-cell embryos developed to blastocysts. The proportion of positive
embryos at the highest titer was 58%, again with a corresponding decrease in mosaicism (Table 1C, Fig. 1C).
Determination of the Timing of Onset of LacZ Activity
Following Injection of AdCMVLacZ/sub360
Mouse embryos. To determine how early LacZ expression could be detected after injection, embryos received
injections at titers that offered an optimal compromise between successful development and reporter gene expression
based on the initial observations listed in Table 1. In the
mouse, this was deemed to be at 2.5 x 103 pfu/100 pl. A
total of 69 embryos received injections and were subsequently divided into three groups, which were examined
for LacZ activity at 30 h, 42 h, or 54 h after injection of
the virus, respectively. None of the first 23 embryos, which
were stained at 30 h and which were all at the 2-cell stage,
expressed the reporter gene. At 42 h, when the embryos
were still at the 2-cell stage, 16 (70%) of the group showed
LacZ activity. One of these positive embryos, which stained
in a single blastomere only, was obviously mosaic. At 54
h, when 21 of the 23 embryos had reached the 4- or 8-cell
stage, all embryos were positive for LacZ, including the
two remaining 2-cell embryos. Interestingly, both of these
were mosaics (Table 2A).
Rat embryos. Rat embryos (75) received injections at a
viral concentration of 2.5 X 104 pfu/100 pl. At 30 h, 23 of
25 embryos in the first group had reached the 2-cell stage,
but none expressed the reporter gene. At 42 h, 24 of 25
embryos were at the 2-cell stage, and only two of these
showed LacZ activity; both were mosaics. In the final
group, which was stained at 54 h, 20 embryos had proceeded to the 4-cell stage. Again, only two of these were
LacZ-positive, and both were mosaics (Table 2B).
Bovine embryos. One-cell zygotes (90) received injections at a viral concentration of 2.5 x 104 pfu/100 pl. After
KUBISCH ET AL.
122
Table 2. Onset of LacZ activity following perivitelline injection of
AdCMVLacZ/sub360 into one-cell mouse, rat, and cow embryos.
Embryo
A) Mouse
B) Rat
C) Cow
Number
injected
23
23
23
25
25
25
30
30
30
Time of
staining
after
injection
30
42
54
30
42
54
30
42
54
h
h
h
h
h
h
h
h
h
Developmental
stage at
staining
LacZ
positive
n (%)
2-cell
2-cell
4-/8-cell
1-/2-cell
2-cell
2-/4-cell
2-/4-cell
4-/8-cell
8-/16-cell
0
16 (70)'
23 (100)b
0
2 (8)
2 (8)
0
6 (20)
7 (23)
Mosaic
n (%)
1 (6)
3 (13)
2 (100)
2 (100)
2 (33)"
6 (86)"
.,.bValues with different superscripts in the same column and species are
significantly different at p < 0.05. Embryos were injected with titers of
2.5 x 10 pfu/100 pl (mouse) and 2.5 x 104 pfu/100 pl (rat, cow).
30 h, 16 of the first group of 30 embryos had cleaved, but
all failed to show evidence of LacZ expression. At 42 h,
22 of the second group had cleaved, and 12 of them had
reached the 8-cell stage. Six (20%) of these embryos expressed the LacZ gene, with two being mosaics (Table 2C).
At 54 h, 21 of the remaining 30 embryos had cleaved at
least once. Of these, seven were positive. The degree of
mosaicism had increased significantly between 42 h and 54
h of culture (p < 0.05, Table 2C).
Effects of Delaying Viral Injection on Reporter
Gene Expression
To determine whether late injections increased the proportion of positive blastomeres, embryos were retrieved at the
1-cell stage and kept in culture until injection. They then received injections of the virus at a concentration of 2.5 x 103
pfu/100 pl (mouse) or 2.5 x 104 pfu/100 pl (rat, cow), were
transferred back into culture, and were kept for a minimum
of 24 h before fixation and staining. To ensure that loss of
ability to infect the embryos was not an artifact of prolonged
in vitro culture, embryos were also retrieved at later stages of
development (morula/blastocyst) from donors (mouse and
rat). These in vivo-reserved embryos likewise received injections and were maintained in culture until staining.
Mouse embryos. Mouse embryos (20) received injections
at the morula stage, i.e., after 3 days in culture. At the same
time, twenty Day 3 embryos were retrieved from a superovuTable 3.
Embryo
A) Mouse
B) Rat
C) Cow
lated donor female. All of the latter embryos were at the
blastocyst stage. All embryos were stained 2 days later (Day
5). By that time, 17 (85%) of the embryos that had received
injections at the morula stage had proceeded to blastocysts,
and six had hatched. All of the in vivo-derived blastocysts
apparently survived the injection, and 18 of them had hatched.
When injection was delayed in this manner, the percentage of embryos that expressed the reporter gene was significantly decreased. Only 35% and 5% expressed the reporter
gene after injection at the morula or blastocyst stage, respectively, compared to 74% when injections had occurred
immediately after zygote retrieval (Tables IA and 3A).
To determine whether the site of injection into blastocysts had any effect either on the degree or pattern of expression of LacZ activity, an additional 40 in vitro-derived
blastocysts received injections of virus either into their
blastocoel cavity or their perivitelline space on Day 4 of
culture. They were stained 2 days later when 15 of the first
group and 12 of the latter group had hatched. Eight (40%)
blastocysts that had received injections into the blastocoelic
cavity expressed the reporter gene. Significantly fewer embryos (2 out of 20 or 10%; p < 0.05) were positive after
the perivitelline space injection. All embryos were mosaics,
and most had only very small patches of blue.
Rat embryos. A total of 120 embryos received injections
in groups of 40 on Day 3 (8-cell stage), Day 4 (morulae),
and Day 5 (blastocysts). An additional 17 blastocysts were
retrieved from a donor 3 days after mating and received
injections into the perivitelline space. All embryos were
fixed and stained at Day 6.
At that time, 18 (45%) of the forty 8-cell embryos that
had received injections on Day 3 had reached the blastocyst
stage. Of the 40 morulae injected on Day 4, 38 were blastocysts on Day 6 (95%). Thirty-seven of 40 in vitro-derived
blastocysts and 14 of 17 in vivo-derived blastocysts survived injection.
Injection of virus at the 8-cell stage resulted in 70% of
the embryos being positive on Day 6. Delaying injection
until the morula stage significantly lowered this percentage
to 33% (p < 0.05). Injection at the blastocyst stage further
decreased the percentage of positive embryos in both
groups (Table 3B). Regardless of the time of injection, all
rat embryos proved to be expression mosaics.
Bovine embryos. Embryos received injections in groups
of 40 on Day 2 (8-cell), on Day 5 (morula), and on Day 7
(blastocyst) post-insemination. The blastocyst group was
further divided into two groups of 20. The embryos in one
Expression of the AdCMCLacZ reporter gene following delayed microinjection.
Developmental stage at injection
Morula
Blastocyst (in vivo)
Blastocyst (in vitro) injected into:
Blastocoel
Perivitelline space
8-cell
Morula
Blastocyst (in vivo)
Blastocyst (in vitro)
8-cell
Morula
Blastocyst, injected into:
Blastocoel
Perivitelline space
Number
injected
Time
stained
LacZ positive
n (%)
Mosaic
n (%)
20
20
40
20
20
40
40
17
40
40
40
after 48 h
after 48 h
7 (35) "'
1 (5)"'
7 (100)
1 (100)
after 48 h
after 48 h
day 6
day 6
day 6
after 24 h
day 9
day 9
8
2
28
13
1
4
40
31
20
20
after 48 h
after 48 h
15 (75)"
12 (60)b
(40)'
(10),'b
(70) d
(33) b
(6)'
(10) c
(100)"
(78)
4 (100)
2 (100)
28 (100)
13 (100)
1 (100)
4 (100)
5 (13),'
17 (55)"
12 (80)" '
8 (67)"
Values with different superscripts in the same column and species are significantly different at p < 0.05.
ADENOVIRUS-MEDIATED GENE TRANSFER
group received injections into the blastocoel, and in the
other, into the perivitelline space. Embryos were maintained
in culture until Day 9, then fixed and stained.
At Day 9, four (10%) of the 8-cell embryos had reached
the blastocyst stage, and 20 were morulae, while the remainder were arrested at earlier developmental stages. All
embryos were positive for LacZ, and only a minority were
mosaics. After injection at the morula stage, 78% of the
embryos expressed the LacZ gene at Day 9; only 55% of
these were mosaics.
No significant differences were noted in the numbers of
positive embryos and the numbers that were mosaic between the blastocysts that had received injections into the
blastocoel and into the perivitelline space. Survival rates
were identical, with one collapsed and 13 hatched blastocysts in each group (Fig. 1D).
DISCUSSION
Our results show that a recombinant human adenovirus
can infect embryos of several nonhuman species. This result is in agreement with a previous report demonstrating
infection of zona-free mouse embryos by a human adenovirus [26]. While the precise mechanism whereby an adenovirus gains entry into cells remains elusive, the viral fiber
capsid may initially bind an as yet unidentified receptor
[27, 28] and then be internalized bound to otav5 integrin
[17, 29]. Although it is unclear whether the virus uses a
similar infection pathway in embryos and whether such embryos express the ctv35 integrin utilized for entry into other
cell types, several other integrins, including ot531 and
av33, have been identified in preimplantation mouse embryos [30]. Both of these integrins recognize the same RGD
(Arg-Gly-Asp) motif in their targets that also mediates the
interaction between adenovirus and avP35. In addition, adenovirus can infect human monocytes which lack otv35 but
which express ctvP3 [31]. Thus, it seems likely that viral
entry into embryos is facilitated by surface integrins. It is
unclear why mouse embryos are infected more efficiently
than rat or cow embryos. The increased efficiency could be
a reflection of the number, type, and onset of expression of
the integrins and receptor proteins required for binding and
internalization of virus.
As viral concentrations were raised, there was increasingly poor embryo development. An effect of the solution
in which the concentrated virus was stored (HEPES-buffered saline containing 10% glycerol) was ruled out by injecting storage solution alone into embryos (data not
shown). While wild-type adenoviruses of most serotypes
have profound effects on host cell DNA and protein synthesis [32, 33], less is known about the consequences of
infection with deficient viruses, particularly on embryonic
cells. In human bronchial epithelial cells, the El deletion
prevented production of the hexon and fiber proteins, but a
subset of cells expressed very high levels of the E2a gene
product DBP (DNA-binding protein) [12]. It is thus possible that embryos are permissive for expression of at least
some viral genes, which may then interfere with embryonic
DNA or protein synthesis. Alternatively, development may
simply be affected because of the presence of a large number of viral genomes in the embryonic nucleus, which could
compete for essential transcription factors. Moreover, some
cytotoxicity has been reported to follow pronuclear injection of DNA, particularly as concentrations are increased
[34-36]. A third possibility is that the LacZ protein itself
has detrimental effects on development [37, 38].
123
Not unexpectedly, the number of embryos and blastomeres that expressed LacZ increased proportionately with
viral concentration. There was also a corresponding decline
in mosaicism as viral titer was raised. Even so, the proportion of positive blastomeres decreased as development
proceeded, and expanded or hatched blastocysts seldom
showed uniform LacZ activity. It has previously been reported that the majority of mouse [39], pig, and cow [9,
10] embryos proved to be expression mosaics after microinjection of LacZ reporter constructs. It was postulated
that such mosaicism could have resulted either from the
selective silencing of reporter genes as development progressed or from persistence of nonintegrated DNA in some
but not all blastomeres. Nonintegrated DNA, particularly if
it is concatamerized and circularized, would presumably
become partitioned with successive cell divisions to a decreasing proportion of blastomeres, resulting in a nonuniform distribution of the DNA. Because adenovirus genomes
are maintained episomally, the virus is an appropriate model to mimic nonintegrated transgenes. Therefore, our observations are entirely consistent with a development-dependent "diluting-out" of the reporter gene.
When 1-cell embryos received injections of virus, reporter gene activity was delayed for at least 42 h, whereas
in cultured cells, adenovirus DNA can reach the nucleus
within 2 h [40]. It is unclear whether these 1-cell embryos
actually internalize the virus immediately or whether there
is a delay until the embryonic genome is activated and the
proper combination of receptor molecules appears on the
surface. Yet even if virus did enter the cell early, the gene
would probably not be efficiently transcribed until the early
2-cell stage in mouse and rat embryos and until the 4- to
8-cell stage in bovine embryos. It is noteworthy that LacZ
expression was readily seen in arrested 1-cell embryos in
all species after several days of culture, a result consistent
with the view that it is the time after fertilization rather
than the developmental stage that governs embryonic genome activation [41-43].
Delaying injection of the virus to later stages of development significantly decreased subsequent LacZ activity in
mouse and rat embryos but had little effect in bovine embryos. Therefore, it seems unlikely that the drop in expression in mouse and rat embryos was a reflection of the lowered capacity of a compressed perivitelline space to retain
virus. Instead, receptor density may fall as embryos advance. Adenovirus uptake in several cell types, including
muscle cells [44], cytotrophoblast [45], and airway epithelial cells [17] declines as they differentiate.
In summary, these results demonstrate that human adenovirus can infect preimplantation embryos of nonhuman
species after they are presented with virus in the perivitelline space. Although high viral titers inhibit embryo development, viral concentrations can be selected that give
excellent expression of a LacZ reporter gene yet have minimal effects on embryo progression to blastocyst. Embryos
can maintain and transcribe episomal DNA at least until the
blastocyst expands and hatches. The mosaic pattern of LacZ
staining observed in embryos as they developed after exposure to different concentrations of virus indicates that
virus can quickly become diluted out. Finally, the efficiency
with which adenovirus infection can be achieved in embryos of certain species, in this case mouse and rat, can fall
as development progresses. Moreover, advancing development and concurrent cellular differentiation may impede the
efficiency with which adenovirus can infect embryos.
124
KUBISCH ET AL.
ACKNOWLEDGMENTS
The authors thank Select Sires, Plains City, OH. for donation of the
bovine semen, and Dr. Don Spiers, Dept. of Animal Sciences, University
of Missouri for supplying the rats.
REFERENCES
I. Gordon JW, Scangos GA, Plotkin DJ, Barbosa JA, Ruddle FH.
Genetic transformation of mouse embryos by microinjection of purified
DNA. Proc Natl Acad Sci USA 1980; 77:7380-7384.
2. Jaenisch R, Jhner D, Nobis P, Simon I, L6hler J, Harbers
K. Grotkopp D. Chromosomal position and activation of retroviral genomes
inserted into the germ line of mice. Cell 1981; 24:519-529.
3. Stewart CL, Schuetze S, Vanek M, Wagner EF Expression of retroviral vectors in transgenic mice obtained by embryo infection. EMBO
J 1987; 6:383-388.
4. Savatier P, Morgenstern J, Beddington RSP Permissiveness to murine
leukemia virus expression during preimplantation and early postimplantation mouse development. Development 1990; 109:655-665.
5. Kim T, Leibfried-Rutledge ML, First NL. Gene transfer in
bovine
blastocysts using replication-defective retroviral vectors packaged
with gibbon ape leukemia virus envelopes. Mol Reprod Dev 1993;
35:105-113.
6. Haskell RE, Bowen RA. Efficient production of transgenic cattle
by
retroviral infection of early embryos. Mol Reprod Dev 1995; 40:386390.
7. Elbrecht A, DeMayo FJ, Tsai M-J, O'Malley BW. Episomal maintenance of a bovine papilloma virus vector in transgenic mice. Mol Cell
Biol 1987; 7:1276-1279,
8. Larson MA, Kubisch HM, Funahashi H, Day BN, Roberts RM.
Expression patterns of two transgenes in murine, porcine and bovine
embryos. Theriogenology 1995; 43:262 (abstract).
9. Kubisch HM, Larson MA, Funahashi H, Day BN, Roberts RM.
Pronuclear visibility, development and transgene expression in IVM/IVFderived porcine embryos. Theriogenology 1995; 44:391-401.
10. Kubisch HM, Hernandez-Ledezma JJ, Larson MA, Sikes JD,
Roberts
RM. Expression of two transgenes in in vitro matured and fertilised
bovine zygotes following DNA microinjection. J Reprod Fertil 1995;
104:133-139.
I1. Lemme E, Eckert J, Carnwath JW, Nieman H. Expression of 6WTKLacZ gene construct in in vitro produced bovine embryos following
microinjection into pronuclei or cytoplasm. Theriogenology 1994; 41:
236 (abstract).
12. Engelhardt JE Yang Y, Stratford-Perricaudet LD, Allen ED, Kozarsky
K, Pericaudet M, Yankaskas JR, Wilson JM. Direct gene transfer
of
human CFTR into human bronchial epithelia of xenografts with
El-deleted adenovirus. Nat Genet 1993; 4:27-34.
13. Davidson BL, Allen ED, Kozarsky KF, Wilson JM, Roessler JA.
A
model system for in vivo gene transfer into the central nervous system
using an adenoviral vector. Nat Genet 1993; 3:219-223.
14. Acsadi G, Jani G, Massie B, Simoneau M, Holland P Blaschuk
K,
Karpati G. A differential efficiency of adenovirus-mediated in vivo
gene transfer into skeletal muscle cells of different maturity. Hum
Mol
Genet 1994; 3:579-584.
15. Kozarsky KE McKinley DR, Austin LL, Raper SE, Stratford-Perricaudet LD, Wilson JM. In vivo correction of low density lipoprotein
receptor deficiency in the Watanabe heritable hyperlipidemic rabbit
with recombinant adenoviruses. J Biol Chem 1994; 269:13695-13702.
16. Engelhardt JE Litzky L, Wilson JM. Prolonged transgene expression
in cotton rat lung with recombinant adenovirus defective in E2a.
Hum
Gene Ther 1994; 5:1217-1229.
17. Goldman MJ, Wilson JM. Expression of avP5 integrin is necessary
for efficient adenovirus-mediated gene transfer in the human airway.
J Virol 1995; 69:5951-5958.
18. Horwitz MS. Adenoviruses and their replication. In: Fields BN, Knipe
DM (eds.), Virology. New York: Raven Press; 1985: 433-476.
19. Chatot CL, Ziomek CA, Bavister BD, Lewis GL, Torres I.
An improved culture medium supports development of random-bred -cell
embryos in vitro. J Reprod Fertil 1989; 86:679-688.
20. Miyoshi K, Funahashi H, Okuda K, Niwa K. Development of rat
onecell embryos in a chemically defined medium: effects of glucose,
phosphate and osmolarity. J Reprod Fertil 1994; 100:21-26.
21. Parrish JJ, Susko-Parrish JL, Leibfried-Rutledge L, Critser ES, Eyestone WH, First NL. Bovine in vitro fertilization with frozen-thawed
semen. Theriogenology 1986; 25:591-601.
22. Hernandez-Ledezma JJ, Villanueva C, Sikes JD, Roberts RM. Effects
of CZB versus Medium 199 and of conditioning with either bovine
oviductal epithelial cells or Buffalo Rat liver cells on the development
of bovine zygotes derived by in vitro maturation-in vitro fertilization
procedures. Theriogenology 1992; 39:1267-1277.
23. Dannenberg AM, Suga M. Histochemical stains for macrophages
in
cell smears and tissue sections: J-galactosidase, acid phosphatase,
nonspecific esterase, succinic dehydrogenase, cytochrome oxidase. In:
Adams D, Edelson P Koren M (eds.), Methods for Studying Mononuclear Phagocytes. New York: Academic Press; 1981: 375-396.
24. Sokal RR, Rohlf FJ. Biometry: The Principles and Practice of Statistics in Biological Research. San Francisco: WH Freeman Co.;
1981:
691-747.
25. SAS. System for Windows. Release 6.11. Cary, NC: Statistical
Analysis System Institute, Inc.; 1995.
26. Tsukui T, Miyake S, Azuma S, Ichise H, Saito I, Toyoda Y.
Gene
transfer and expression in mouse preimplantation embryos by recombinant adenovirus vector. Mol Reprod Dev 1995; 42:291-297.
27. Philipson L, Lonberg-Holm K, Petterson U. Virus-receptor interaction
in an adenovirus system. J Virol 1968; 2:1064-1075.
28. Defer C, Belin MT, Caillet-Boudin ML, Boulanger P. Human adenovirus-host cell interactions: comparative study with members of subgroups B and C. J Virol 1990; 64:3661-3673.
29. Wickman TJ, Filardo EJ, Cheresh DA, Nemerow GR. Integrin
vl5
selectively promotes adenovirus mediated cell membrane permeabilization. J Cell Biol 1994; 127:257-264.
30. Sutherland AE, Calarco PG, Damsky CH. Developmental regulation
of integrin expression at the time of implantation in the mouse embryo. Development 1993; 119:1175-1186.
31. Huang S, Endo RI, Nemerow GR. Upregulation of integrins
iav33 and
cvv35 on human monocytes and T lymphocytes facilitates adenovirusmediated gene delivery. Virol 1995; 69:2257-2263.
32. Hodge LD, Scharff MD. Effect of adenovirus on host cell DNA
synthesis in synchronized cells. Virology 1969; 37:554-564.
33. Pina M, Green M. Biochemical studies on adenovirus multiplication.
XIV. Macromolecule and enzyme synthesis in cells replicating oncogenic and non-oncogenic human adenoviruses. Virology 1969;
38:
573-586.
34. Gordon JW, Ruddle FH. Integration and stable germ line transmission
of gene injected into mouse pronuclei. Science 1981; 214:1244-1246.
35. Brinster RL, Chen HY, Trumbauer ME, Yagle MK, Palmiter RD. Factors affecting the efficiency of introducing foreign DNA into mice
by
microinjecting eggs. Proc Natl Acad Sci USA 1985; 82:4438-4442.
36. Gagne M, Pothier F, Sirard MA. Foreign gene expression in activated
oocytes and bovine embryos following pAGS-LacZ plasmid microinjection. Theriogenology 1993; 39:223 (abstract).
37. Kimura S, Niwa H, Moriyama M, Araki K, Abe K, Miike T,
Yamamura K. Improvement of germ line transmission by targeting -galactosidase to nuclei in transgenic mice. Dev Growth Differ 1994; 36:
521-527.
38. Paldi A, Deltour L, Jami J. Cis effect of lacZ sequences in transgenic
mice. Transgenic Res 1993; 2:325-329.
39. Takeda S, Toyoda Y. Expression of SV40-LacZ gene in mouse preimplantation embryos after pronuclear microinjection. Mol Reprod Dev
1991; 30:90-94.
40. Chardonnet Y, Dales S. Early events in the interaction of adenoviruses
with Hela cells. III. Relationship between ATPase activity in nuclear
envelopes and transfer of core material. A hypothesis. Virology 1972;
48:342-359.
41. Lathan KE, Solter D, Schultz RM. Acquisition of a transcriptionally
permissive state during the -cell stage of mouse embryogenesis. Dev
Biol 1992; 149:457-462.
42. Veinet M, Bonnerot C, Borand P, Nicolas JE Changes in permissiveness for the expression of microinjected DNA during the first cleavages of mouse embryos. Mech Dev 1992; 36:129-139.
43. Ram PT, Schultz RM. Reporter gene expression in G2 of the
I-cell
mouse embryo. Dev Biol 1995; 156:552-556.
44. Acsadi G, Jani G, Massie B, Simoneau M, Holland P Blaschuk
K,
Karpati G. A differential efficiency of adenovirus-mediated in vivo
gene transfer into skeletal muscle cells of different maturity. Hum Mol
Genet 1994; 3:579-584.
45. MacCalman CD, Furth EE, Omigbodun A, Kozarsky KF, Coutifaris
C, Strauss JE Differentiation-dependent transduction of human trophoblast cells by recombinant adenovirus. Biol Reprod 1996; 54:682691.