1022-7954/03/3906- $25.00 © 2003
Russian Journal of Genetics, Vol. 39, No. 6, 2003, pp. 627–629. Translated from Genetika, Vol. 39, No. 6, 2003, pp. 758–761.
Original Russian Text Copyright © 2003 by Andreeva, Sleptsova, Grigorenko, Gavrushkin, Kuznetsov.
In some cases, sperm cells (SCs) can uptake foreign
DNA from the outer medium . Bound DNA is
always found in the subacrosomal segment of the SC
head, where its nucleus is located, and may integrate
into the SC genome , express in the embryo after fer-
tilization [3–5], and be transmitted to the next genera-
tion . More often, however, the foreign DNA is
degraded, undergoes structural rearrangements, loses
its functional activity, and is eliminated from the
genome [7, 8]. Some protein factors (30- to 35-kDa
proteins, CD4, and others) have been found to ensure
the binding of foreign DNA and its penetration into SCs
. The amount of foreign DNA that SC can transfer
attains 10% of the amount of its own DNA .
To demonstrate the nontrivial phenomenon of the
sperm-mediated transfer of foreign genetic informa-
tion, we used the efﬁcient method of SC electrotrans-
fection. After fertilization, the expression of reporter
DNA in loach embryos and larvae was estimated.
MATERIALS AND METHODS
Object of the study.
The object of the study was the
L.; family Cobitidae, order
Cypriniformes, superorder Teleostei). Loach males and
females were caught in a natural population (the Rya-
zan’ oblast) in November and December and stored
separately in a refrigerator at 4–6
C. Mature ova were
obtained 40–46 h after choriogonin injections at doses
varying from 100 to 250 IU depending on the season
and the size of the female . Mature SCs were
obtained from the testes of sacriﬁced males.
We used the commercial plasmid pcDNA3-
(Invitrogen, United States) containing the pro-
moter of human cytomegalovirus (CMV).
Sperm was squeezed out of a 5-mm
long fragment of the testis in a drop of sterile tap water
with an electric conductance about 4 k
/cm. A 10-
aliquot of the resultant suspension was transferred into
l drop and stirred by pipetting. Then, 10
this suspension was passed through another (identical)
drop, and 1/10 of the resultant suspension was trans-
ferred to a water drop that was identical to the preced-
ing ones except that it contained 5
g of DNA. The drop
was stirred thoroughly. Then, we placed 100
l of the
SC suspension into a cooled electrotransfection cuvette
and passed an electric discharge (V = 500 V, R = 150
C = 20
F) through it (the device was from Bioimpul’s,
NPO Biopribor, Russia). The operations were per-
formed in Petri dishes on ice as quickly as possible.
Three control groups of embryos were used: embryos
resulting from natural fertilization (C1) and embryos
resulting from eggs fertilization by either SCs subjected
to an electric discharge (C2) or SCs transfected with
plasmid pcDNA3 (mock analysis; C3).
Fertilization and incubation.
Fifty microliters of the
suspension of transfected SCs and 1 ml of water were
added to approximately 100 dry eggs and shaken gen-
tly. Three minutes later, the eggs were washed with
water, and fresh water was added to a ﬁnal volume of
10 ml. Fertilized eggs were incubated in large vessels at
room temperature for ﬁve days. We used settled tap
water containing antibiotics (ampicillin and streptomy-
g/ml of each). Intravital monitoring of embry-
onic development and comparison of control and
experimental (both live and ﬁxed) embryos were per-
formed with the use of an MBS-9 binocular microscope
and an Opton inverted microscope. The developmental
stages were identiﬁed according to the reference tables
of normal development .
Estimation of expression.
The embryos were ﬁxed
with 2.5% glutaraldehyde, washed with phosphate
Loach Sperm Cells Transfer Foreign DNA
Expressed in Early Development
L. E. Andreeva
, L. A. Sleptsova
, A. P. Grigorenko
A. V. Gavrushkin
, and A. V. Kuznetsov
Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, 123182 Russia;
fax: (095)196-02-21; e-mail: firstname.lastname@example.org
Moscow State University, Moscow, 119899 Russia
Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow oblast, 142292 Russia
Institute of Professional Pedagogy, Magdeburg, 39104 Germany; e-mail: email@example.com
Received April 26, 2002; in ﬁnal form, July 14, 2002
—The transfer of plasmid pcDNA3
by electrotrasfected sperm cells into loach (
L.) ova has been studied. The
gene has been found to express in 3- to 5-day-old prelarvae.