ISSN 0026-8933, Molecular Biology, 2017, Vol. 51, No. 4, pp. 592–595. © Pleiades Publishing, Inc., 2017.
Original Russian Text © M.V. Tikhonov, O.G. Maksimenko, P.G. Georgiev, I.V. Korobko, 2017, published in Molekulyarnaya Biologiya, 2017, Vol. 51, No. 4, pp. 671–676.
Optimal Artificial Mini-Introns for Transgenic Expression
in the Cells of Mice and Hamsters
M. V. Tikhonov*, O. G. Maksimenko, P. G. Georgiev, and I. V. Korobko
Institute of Gene Biology, Russian Academy of Sciences, Moscow, 119334 Russia
Received July 24, 2016; in final form, October 24, 2016
Abstract⎯Introns can frequently enhance transgene expression, and sometimes they are absolutely substan-
tial. Based on an analysis of murine genes, in which mRNA does not have alternative splicing, a universal
design of the efficiently spliced artificial introns of small sizes has been proposed. These introns are shown to
be eff iciently spliced in CHO cells from hamster ovaries. The proposed strategy can be used to include introns
in cDNA, which would elevate the production of recombinant proteins in cell culture, as well as in transgenic
Keywords: artificial intron, cDNA, expression
Introns can frequently enhance transgene expres-
sion, and sometimes they are absolutely substantial
[1‒5]. This suggests the inclusion of introns in expres-
sion constructs designed for production of recombi-
nant proteins. The CHO cell line derived from the
hamster ovaries is conventionally used to produce
recombinant protein and is also relevant in the use of
introns . Introns can be of significant importance in
transgenic animals due to the substantial increase in
transgene expression (see, e.g., ). For this reason,
complete genomic sequences of genes (not intronless
cDNA copies) are preferable for transgenesis. How-
ever, large DNA sequences in turn lead to genetic con-
structs of significant sizes, which makes their creation
and manipulation difficult.
The intron-dependent enhancement of gene
expression can be associated with enhancer sequences
located in introns. However, aside from these
enhancers, the intron splicing affects gene expression
at the posttranscriptional level .This is another evi-
dence in favor of inclusion of both endogenic and
exogenic introns in transgenes. In fact, several vectors,
for example pCI (Promega, United States), carry
mini-introns in their transgenes, which were obtained
by the elimination of the long mid-regions of introns
. However, this approach requires an individual
analysis in each specific case . Moreover, if several
introns must be included, alternative splicing can
complicate the construction task. All of this led to the
development of the standard strategy in the construc-
tion of artificial mini-introns with efficient splicing,
which simplifies their insertion into target cDNA.
Several such introns located in the same transcript can
be used without a risk of the alternative splicing.
The minimum set of intron structural components
required for splicing includes the donor and acceptor
sites, branch point, and polypyrimidine region located
between the branch point and the acceptor site .
An artificial mini-intron has to include the entire set
with the optimal location of its components. This
strategy allowed one to create an artificial intron that
was efficiently spliced in Shizophillum commune. The
transgene with this intron was expressed at a high level,
which would be impossible if there was no intron .
Since consensus sequences of splicing sites can vary in
different organisms , the choice of a model is
important. The house mouse (Mus musculus) was cho-
sen in this study because it is a basic model for trans-
genesis and is phylogenetically close to the Chinese
hamster (Cricetulus griseus), the cells of which gave
rise to the biotechnologically important CHO cell line
and its derivatives.
Plasmids. To obtain the basic version of the artifi-
cial intron two oligonucleotides of 5'-gcactcgagaaggta-
included 45 nt of the intron 5'-region, and 5'-gtatct-
which was complementary to the intron 3'-region,
were annealed by the 15 complementary nucleotides
and filled in with Pfu DNA polymerase (Thermo
Fisher Scientific, United States). The resulting DNA
Abbreviation: EGFP, enhanced green fluorescent protein.
MOLECULAR CELL BIOLOGY