1062-3604/04/3505- © 2004
Russian Journal of Developmental Biology, Vol. 35, No. 5, 2004, pp. 316–318. Translated from Ontogenez, Vol. 35, No. 5, 2004, pp. 387–389.
Original Russian Text Copyright © 2004 by Shishimorova, Ivanova, Ryabykh.
Improvement of the methods of transgenic technol-
ogy is aimed at elevation of the frequency of integration
of foreign genes and their efﬁcient expression in a
genetically modiﬁed organism. The experimental data
suggest that the presently most widespread method of
microinjection of a gene engineering construct in the
pronucleus of a zygote does not provide for a high efﬁ-
ciency of transgenesis in animals (Persuy
Aguerre and Castro-Palomino, 1998).
The current concepts of cytogenetics suggest that
foreign DNA is incorporated at the sites of natural
breaks of the host chromosomal DNA at the early
embryonic stages (Bishop and Smith, 1989). Simulta-
neous injection of recombinant DNA and site-speciﬁc
endonucleases in human cells stimulates homologous
recombinations between foreign DNA and chromo-
somal host DNA (Brenneman
, 1996). As a result,
it has been proposed that combined microinjection of a
gene constructs and endonuclease, which was used to
separate the given gene construct from the vector, in the
pronucleus of a zygote enhances the cleaving of the
recipient chromosomal DNA with the formation of
adhesive ends complementary to those of the injected
gene engineering construct.
The aim of this work was to study the effects of
combined microinjection of a gene engineering con-
struct and restrictase in the pronucleus of zygotes on
preimplantation development of mouse embryos
MATERIALS AND METHODS
Studies were carried on hybrid mice (CBA
. Zygotes were extracted by washing ovi-
ducts with a medium M2 enriched by 0.4% BSA
(Sigma, USA) within 7–8 h after mating.
The gene construct
, which contains the
structural gene of a human factor stimulating the for-
mation of granulocyte colonies (
with regulatory sequences of the gene
casein, was separated from the vector by restriction of
the recombinant plasmid with site-speciﬁc endonu-
. The isolated DNA fragment was dissolved
in buffer TE to a concentration of 5–6 ng/ml. Endonu-
and corresponding 10-fold buffer Orange
(Fermentas, USA) were introduced in a solution with
the gene construct directly before microinjection.
Microinjection was performed on a manipulator
equipped with an inverted Nicon microscope (Japan)
and differentiation-interference Nomarski contrast.
Embryos were cultivated in medium M16 comple-
mented with 0.5% BSA in Petri dishes under mineral
oil (Sigma, USA) in a gas phase containing 5%
. The viability of microinjected zygotes was
assessed by their capacity to develop to the blastocyst
stage with subsequent hatching from zona pellucida.
The data obtained were processed statistically using
Influence of Combined Microinjection of Gene Engineering
Construct and Endonuclease on Preimplantation Development
of Mouse Embryos
M. S. Shishimorova, L. B. Ivanova, and V. P. Ryabykh
National Research Institute of Physiology, Biochemistry, and Feeding
of Farm Animals, Borovsk, Kaluga oblast, 249013 Russia
Received June 25, 2003; in ﬁnal form, October 29, 2003
—We studied the inﬂuence of combined microinjection of a gene engineering construct and site-spe-
in the pronucleus on preimplantation development of (CBA
. The rate of survival of the embryos was estimated according to their capacity to develop until
the blastocyst stage and hatch from zona pellucida. The results obtained suggest that the microinjection of exog-
enous DNA jointly with endonuclease
at concentrations from 0.1 to 0.01 U/
l decreased reliably the rate
of survival, as compared to the control (
< 0.05 and
< 0.01, respectively). However, a decrease of endonu-
concentration in the injection mixture to 0.01 U/
l enhanced the capacity of mouse embryos to
develop until the blastocyst stage and hatch from zona pellucida, as compared to the embryos microinjected
with exogenous DNA and endonuclease
at a higher concentration.
: gene engineering construct, site-speciﬁc endonuclease, microinjection, pronucleus.