ISSN 1062-3604, Russian Journal of Developmental Biology, 2008, Vol. 39, No. 3, pp. 168–171. © Pleiades Publishing, Inc., 2008.
Original Russian Text © M.V. Glazkov, S.F. Drozd, P.K. Golovatenko-Abramov, M.Yu. Martynova, A.P. Nesterova, 2008, published in Ontogenez, 2008, Vol. 39, No. 3, pp. 208–211.
The manipulations with zygotes during transgene
introduction into the male pronucleus (as well as the
proper introduction of foreign DNA) substantially
decreases the embryonic viability and gives rise to
numerous nonviable offspring with various develop-
mental abnormalities (Brinster et al., 1985; Andreeva
and Serova, 1992). Among many factors underlying
this phenomenon (Kadulin et al., 2006), we consider
only one, the structure of the genetic construct used to
transform the zygote.
MATERIALS AND METHODS
The p1446 plasmid carrying the
the control of the human
-globin promoter and the
800-bp regulatory region of the
was kindly provided by S. Nonchev (Universite Joseph
Fourier, Grenoble, France).
Experiments were carried out on CBA (female) and
C57BL6 (male) mice at the age of 2–2.5 months.
Production of mice with known gestational age.
Mice (at the age of 8–10 weeks) were induced to ovu-
lation by intraperitoneal administration of serum (Folli-
gon, Intervet, United Kingdom) and chorionic (Chorulon,
Intervet, United Kingdom) gonadotropins (5 and 10 IU of
each hormone per mouse with an interval of 46 h). Then
the females were exposed to males overnight. The day
of the vaginal blood detection (in the morning) was
considered as the ﬁrst gestation day.
Isolation of oocytes from the oviducts.
oocytes were released from follicular cells in Whitten’s
medium with BSA (Abramezuk et al., 1977) supple-
mented with 0.1% hyaluronidase (Sigma, United
States) (Hogan et al., 1986). The embryos were thor-
oughly washed from hyaluronidase in drops of Whit-
ten’s medium with BSA by transferring them from drop
to drop using a glass pipette with the inner diameter of
m. All manipulations with the early mouse
embryos were performed in Dulbecco’s PBS and in
Transformation of zygotes and in vitro culture of
TE buffer (1–2 pl; 0.1 mM EDTA and 10 mM
Tris-HCl, pH 7.4; Fluka, Germany) containing 1500–
3000 copies of the linearized transgene was injected
into the male pronucleus using a Narichige microman-
ipulator (Japan) under a Biolam microscope (Russia).
The preimplantation embryos were cultured to the blas-
tocyst stage in glass ﬂasks in 500
l of Whitten’s
C under 5% CO
and 90% N
Assay for the survival of preimplantation mouse
The number of viable embryos was visually
determined immediately before transferring the fertil-
ized eggs to a culture ﬂask and at the end of the culture
period (day 4). In addition, the number of normal and
abnormal embryos that have not reached the late
morula or blastocyst stage as well as the number of
abnormal morulae, blastocysts, and dead embryos were
counted on day 4 of culture.
Production of 9.5–11.5-day embryos.
zygotes were transplanted into the oviduct funnel of
pseudopregnant females (CBA
by mating with vasectomized males on the previous
Pregnant females were killed by cervical dislocation
9.5–11.5 days after transplantation and the embryos
Survival of Transgenic Mouse Embryos Carrying Different
M. V. Glazkov
, S. F. Drozd
, P. K. Golovatenko-Abramov
M. Yu. Martynova
, and A. P. Nesterova
Kol’tsov Institute of Developmental Biology, Russian Academy of Sciences, ul. Vavilova 26, Moscow, 119991 Russia
Vavilov Institute of General Genetics, Russian Academy of Sciences, ul. Gubkina 3, Moscow, 119991 Russia
Received July 4, 2007; in ﬁnal form, September 26, 2007
—The survival of transgenic mouse embryos was studied as a function of the transgene structure. The
data obtained indicate that the introduction of a chromosomal DNA fragment providing for the anchoring of
interphase chromosomes on the nuclear envelope increases the efﬁciency of transgenesis in mice threefold due
to their increased viability.
: transgenesis, embryonic viability, compartmentalization of genes in the nucleus.