3. Results
3.1. Effect of EcoRI microinjected into the pronucleus on embryo surÕiÕal To determine the optimal amount of the enzyme for the co-injection, we injected
EcoRI alone in varying amounts ranging from 10
y9
Urnucleus to 10
y5
rnucleus, and examined the survival rate of the embryos. The buffer used for dissolving DNA was
injected as the control. Table 1 shows the number of embryos that survived and developed in vitro afte rmicroinjection of various amounts of EcoRI into the pronucleus
Ž .
of zygotes. A sufficiently high proportion 71 of the eggs given the injection of the buffer alone developed into the morularblastocyst stage in vitro. Approximately 70 of
the embryos given the injection of 10
y9
Urnucleus developed into the morularblasto- cyst stage. Injection of 10
y6
Urnucleus of EcoRI resulted in a remarkable reduction in the viability of the injected eggs, though 12 of the injected embryos developed to the
Ž .
y5
four-cell stage data not shown . The concentrations more than 10 Urnucleus did not
result in any viable embryos. Injections of amounts of 5 = 10
y8
and 10
y7
Urnucleus, resulted in survival rates of 60.6 and 48.5 of the embryos, respectively. There was a
good linear correlation between the amount of the enzyme injected and the mean survival rate of the injected eggs. We used 5 = 10
y8
Urnucleus in the subsequent series of experiments in which the enzyme was co-injected with DNA because this concentra-
Ž .
tion gave reasonable survival rate 60.6 before drastic reduction. 3.2. Effects of the co-injection of DNA constructs with the restriction endonuclease on
the integration rate of foreign genes The results of co-injecting DNA constructs with restriction endonuclease are shown
in Table 2. In this series, each of the two gene constructs was dissolved in the injection
Table 1 In vitro development of mouse zygotes that were given pronuclear injections of various amounts of EcoRI
Ž . Treatment
No. of No. of
No. of developed embryos
a
trials eggs used
Morulae Blastocyst
Total Ž
. Ž
. Ž
. Non-injection
9 294
7 2.3 275 93.5
282 95.9
b
Ž .
Ž .
Ž .
Injection of buffer 2
107 15 14.0
61 57.0 76 71.0
y9
Ž .
Ž .
Ž .
Amounts of injected 10
2 63
8 12.7 36 57.1
44 69.8
y8
Ž .
Ž .
Ž .
Ž .
enzyme Ur2 plr nucleus 10
3 170
12 7.1 108 63.5
120 70.6
y8
Ž .
Ž .
Ž .
5=10 2
61 8 13.8
29 50.0 37 60.6
y7
Ž .
Ž .
Ž .
10 3
134 14 10.4
51 38.1 65 48.5
y7
Ž .
Ž .
Ž .
5=10 2
75 4 5.4
30 40.5 34 45.9
y6
Ž .
Ž .
Ž .
10 2
45 6 13.3
5 11.1 11 24.4
y6
Ž . Ž
. Ž
. 5=10
2 47
0 0 2 4.3
2 4.3
y5 c
Ž . Ž .
Ž . 10
1 25
0 0 0 0
0 0
a
The embryos did not develop beyond morula.
b
Ž .
10 mM Tris–HCl pH 8.0 , 1 mM EDTA.
c
Ž .
Host genome DNA 3 pg in the nucleus is expected to be completely digested.
B.B. Seo
et al.
r Animal
Reproduction
Science 63
2000 113
– 122
118
Table 2 Effects of the co-injection of foreign genes with or without EcoRI into the pronucleus of mouse zygotes on transgenesis in single embryos and pups born
Each value of gene injections with or without the enzyme includes the results of two or three trials. Injected foreign genes
Gene with EcoRI Gene alone
Overall efficiency mWAPrhGH
Human Agrb-globin mWAPrhGH
Human Agrb-globin Gene with EcoRI
Gene alone
a
Ž . Ž
. Ž
. Ž
. Ž
. Ž
. Ž
. No. of transgenic embryos
6r36 16.7 4r21 19.0
2r21 9.5 3r23 13.0
10r57 17.5 5r44 11.4
b
Ž . Ž .
Ž .
Ž . Ž .
Ž .
Ž . No. of transgenic pups
0r5 0 2r5 40.0
0r6 0 0r5 0
2r10 20.0 0r11 0
c c
Ž . Ž
. Ž
. Ž
. Ž
. Ž
. Ž
. Overall of transgenesis
6r41 14.6 6r26 23.1
2r27 7.4 3r28 10.7
12r67 17.9
5r55 9.1
a
Ž .
DNA extracted from the single late–stage embryo was digested with DpnI and Bal31, and was analyzed by PCR Soe et al., 1997 .
b
DNA extracted from tail tip of pups born was analyzed by PCR.
c
Ž
2
. 0.1- P - 0.2
x test .
Ž .
Ž
y8
. buffer 1 mgrml containing EcoRI equivalent to 5 = 10
Urnucleus and injected into a zygote pronucleus. The human Agrb-globin fusion gene has the ClalrSal l
restriction ends, but does not have EcoRI site within its construct. The embryos that survived the injection were cultured in vitro until they developed into the morular
blastocyst stage. Fig. 1 shows representative photographs of the agarose-gel elec- trophoresis of PCR products of DNA extracted from transgenic single embryos follow-
ing the co-injection of DNA with EcoRI. The embryos that integrated the foreign gene
Ž .
were identified by the Dpnl–Bal31 digestion method described by Seo et al. 1997 , while the transgenic pups were identified by the standard PCR method. Co-injection of
Fig. 1. Representative agarose–gel electrophoretograms of PCR products using the DNA extracted from the single blastocysts as the templates after a combined digestion with DpnI endonuclease and Bal31 exonuclease
Ž .
see Materials and Methods . Arrows indicate the positions of PCR products amplified by the specific primers. mWAPrhGH and human
A
grb-globin indicate the gene constructs that injected to the pronucleus of zygotes, alone or with EcoRI. M, molecular weight markers. The same PCR products were detected by analysis of
Ž .
DNAs extracted from pups born data not shown .
the genes with EcoRI appeared to have resulted in the elevation of the rate of integration in the embryos examined: the overall efficiency of the embryos that
integrated the foreign genes was 17.5 for the co-injection group and 11.4 for the DNA alone group. Seven of 12 recipients with the transfer of the injected embryos gave
birth to 21 pups. The overall efficiency of transgenesis in pups with the co-injection was 20.0, while no transgenic pups were born when DNA was injected alone. The overall
efficiency of the integration of foreign DNA in the embryos and live-born pups obtained by the present co-injection procedures was 17.9 compared with 9.1 obtained by the
injection of DNA alone. When the DNA injection with or without EcoRI was compared regarding overall efficiency of transgenesis, the co-injection group was better than the
control group, although the difference between the two groups, was not statistically
Ž Ž
2
.. significant 0.1 - P - 0.2
x test .
4. Discussion
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