An Alternative Method to Facilitate cDNA Cloning for Expression Studies in Mammalian Cells by Introducing Positive Blue White Selection in Vaccinia Topoisomerase I-Mediated Recombination
September
An Alternative Method to Facilitate cDNA Cloning for Expression Studies in Mammalian Cells by Introducing Positive Blue White Selection in Vaccinia Topoisomerase I-Mediated Recombination
Hiroshi Udo 0 1
0 Department of Biology, Graduate School of Science, Kyushu University , Fukuoka , Japan
1 Editor: Yuntao Wu, George Mason University , UNITED STATES
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One of the most basic techniques in biomedical research is cDNA cloning for expression
studies in mammalian cells. Vaccinia topoisomerase I-mediated cloning (TOPO cloning by
Invitrogen) allows fast and efficient recombination of PCR-amplified DNAs. Among TOPO
vectors, a pcDNA3.1 directional cloning vector is particularly convenient, since it can be
used for expression analysis immediately after cloning. However, I found that the cloning
efficiency was reduced when RT-PCR products were used as inserts (about one-quarter).
Since TOPO vectors accept any PCR products, contaminating fragments in the insert DNA
create negative clones. Therefore, I designed a new mammalian expression vector
enabling positive blue white selection in Vaccinia topoisomerase I–mediated cloning. The
method utilized a short nontoxic LacZα peptide as a linker for GFP fusion. When cDNAs
were properly inserted into the vector, minimal expression of the fusion proteins in E. coli
(harboring lacZΔM15) resulted in formation of blue colonies on X-gal plates. This method
improved both cloning efficiency (75%) and directional cloning (99%) by distinguishing
some of the negative clones having non-cording sequences, since these inserts often
disturbed translation of lacZα. Recombinant plasmids were directly applied to expression
studies using GFP as a reporter. Utilization of the P2A peptide allowed for separate expression
of GFP. In addition, the preparation of Vaccinia topoisomerase I-linked vectors was
streamlined, which consisted of successive enzymatic reactions with a single precipitation step,
completing in 3 hr. The arrangement of unique restriction sites enabled further modification
of vector components for specific applications. This system provides an alternative method
for cDNA cloning and expression in mammalian cells.
Vaccinia topoisomerase I-mediated cloning, known as TOPO cloning by Invitrogen, has been
widely used in biological and medical research. It was originally developed by Shuman [1],
which revolutionized the way to clone PCR-amplified DNAs. It is quick and efficient without
relying on restriction enzymes and DNA ligases. Since the cloning procedure is very simple,
this method is suitable for large scale cloning as well [2].
The cloning method is based on unique properties of Vaccinia topoisomerase I [3]. The
enzyme recognizes a specific pentapyrimidine sequence of 5’-(C/T)CCTT#-3’ and hydrolyzes
the phosphodiester bond at the 3’-end of its recognition site [4]. Upon cleavage, a covalent
bond is formed between DNA and the enzyme (3’-phosphate of the DNA and tyrosine 274 of
the enzyme), and the nicked strand is relaxed in the mean time. The phosphor-tyrosyl bond
energy is then utilized to relegate to the 5’-hydroxyl end of the cleaved strand, and the enzyme
is released from the DNA after reaction. When the enzyme reacts with a linear double-stranded
DNA having its recognition site near the 3’-end, a short terminal fragment is released from the
nicked strand, and the enzyme remains covalently bound to the DNA. This enzyme-DNA
complex is stable, and is able to recombine with an exogenous DNA having a 5’-hydroxyl end.
In the original cloning procedure, insert DNAs were first reacted with Vaccinia
topoisomerase I, and the resulting enzyme-insert DNA complex was then added to the dephosphorylated
vector DNA [1]. This procedure was later modified to a well-known format, in which the
enzyme was linked with the vector DNA (TOPO vector) to accept insert DNAs having
5’hydroxyl ends. This is particularly suitable for cloning of PCR-amplified DNAs, since
chemically synthesized primers have a 5’-hydroxyl end. If TOPO vectors are ready, recombination
can be done by simply adding PCR products.
A variety of TOPO vectors are commercially available today. However, the most popular
applications may be cDNA cloning for expression studies in mammalian cells. Since expression
vectors require cDNAs to be inserted in the correct orientation, directional TOPO cloning
vectors are suitable (such as a pcDNA3.1 directional TOPO cloning vector from Invitrogen).
Oriented insertion was facilitated by the inclusion of four bases “CACC” (corresponding to a
portion of Kozak sequence [5]) at the 5’-end of the insert DNA and also at the cloning site of
the vector. Furthermore, pcDNA3.1 allows expression of cloned cDNAs in mammalian cells
under control of a CMV promoter [6]. This is time and cost-efficient since conventional
strategies involve two steps, cDNA cloning into a cloning vector and subcloning into an expression
vector.
I attempted to clone mouse cDNAs using the pcDNA3.1 directional (...truncated)