1022-7954/05/4103- © 2005 Pleiades Publishing, Inc.
Russian Journal of Genetics, Vol. 41, No. 3, 2005, pp. 227–232. Translated from Genetika, Vol. 41, No. 3, 2005, pp. 299–306.
Original Russian Text Copyright © 2005 by Zakharova, Pryzhkova, Kibardin, Ermolkevich, Kadulin, Gnuchev, Kiselev.
The progress achieved in molecular biology and
bioengineering in recent decades has made it possible
to construct transgenic animals. To date, construction,
analysis, and use of transgenic animals in research and
practice have been reported in many articles [1–5]. The
techniques for constructing transgenic animals include
microinjection of DNA into a pronucleus of a zygote,
infection of preimplantation embryos with recombinant
retroviruses, and transfection of embryonic stem cells
(ESC) with recombinant DNA with subsequent ESC
introduction into blastocysts.
The objectives of this work were to obtain genetic
constructs and to construct transgenic animals produc-
ing human lactoferrin in milk. Lactoferrin plays numer-
ous metabolic and regulatory roles, the major of which
is iron binding and transport. Lactoferrin suppresses the
growth of potentially pathogenic bacteria of several
groups in the mammary gland of the mother and in the
gastrointestinal tract of the child . We have previ-
ously considered the functions of human lactoferrin
and the possibilities of its use in therapy and disease
prevention . A natural source of human lactoferrin is
breast milk (1–5 g/l); in addition, lactoferrin has been
found in neutrophils . Several preparations contain-
ing human milk lactoferrin have been developed and
patented [9, 10]. However, such preparations are pro-
duced on a small scale because of the limited amount of
donor milk. The demands for lactoferrin as a medicinal
agent and a component of baby food for bottle feeding
is far greater than can be satisﬁed with the available nat-
ural resources. Hence, it is necessary to produce lacto-
ferrin artiﬁcially. A promising method is to express
recombinant human lactoferrin in the mammary gland
of transgenic animals. For this purpose, two genetic
constructs, LK1 and G8, were obtained in our labora-
tory. The constructs each contained the human lactofer-
rin gene under the control of regulatory regions of the
casein gene. The regulatory regions proved
to be efﬁcient in our previous experiments on expres-
sion of endostatin in the mammary gland of transgenic
mice . Microinjecting recombinant DNA into a
zygote pronucleus, we obtained several transgenic mice
carrying the constructs. In addition, the construct LK1
was used to transfect mouse ESC. Transcription of the
transgene and splicing of the recombinant mRNA were
studied in the mammary gland of transgenic mice and
in transfected ESC lines.
MATERIALS AND METHODS
Enzymes and reagents.
We used enzymes from Fer-
mentas (Lithuania), Promega (United States), and
Gibco (United States) and reagents from Sigma (United
States), Invitrogen (United States), Amersham (United
States), Qiagen (Germany), and Merck (Germany).
Autoradiography was performed with a Kodak BioMax
MS ﬁlm (United Kingdom) with intensifying screens.
Human lactoferrin cDNA.
To synthesize the lactof-
errin cDNA, RNA was isolated from human neutro-
phils. Reverse transcription was carried out with a
SuperScript II RNase H–reverse transcriptase kit (Invit-
rogen) and random hexamer primer pdNx6 (Amer-
sham) as recommended by the manufacturers. Ampliﬁ-
cation of the cDNA was performed using an eLONGase
enzyme mix kit (Invitrogen) and oligonucleotides LAC1
(5'-gtttgccaagtcgcctccaga-3') and LAC14 (5'-ggcagtgaatg-
gctgaggctt-3') as recommended by the manufacturer.
Transcription and mRNA Splicing of the Human Lactoferrin Gene
Controlled by the Regulatory Region of the Bovine
Casein Gene in the Mammary Gland of Transgenic Mice
and in Mouse Embryonic Stem Cells
E. S. Zakharova, M. V. Pryzhkova, A. V. Kibardin, T. G. Ermolkevich,
S. G. Kadulin, N. V. Gnuchev, and S. L. Kiselev
Institute of Gene Biology, Russian Academy of Sciences, Moscow, 117334 Russia; fax: (095)135-41-05; e-mail: email@example.com
Received April 6, 2004
—The regulatory region of the bovine
casein gene was used to obtain two genetic constructs for
expression of human lactoferrin in the mammary gland of transgenic animals. Several transfected mouse
embryonic stem cell (ESC) lines and primary transgenic mice were generated with these constructs. Recombi-
nant lactoferrin was not detected in milk of transgenic mice by Western blotting. However, a recombinant tran-
script was found in RNAs isolated from mammary glands of transgenic females during lactation and from trans-
fected ESC lines.