Transcription from plasmid expression vectors is increased up to 14-fold when plasmids are transfected as concatemers
Transcription from plasmid expression vectors is increased up to 14-fold when plasmids are transfected as concatemers
Patrick
Leahy*
,
Gordon G.
Carmichael
1
and
Edward F.
Rossomando
Department of BioStructure and Function and
1
Department of Microbiology, University of Connecticut Health Center,
Farmington
, CT 06030,
USA
Received August 19, 1996;
Revised and Accepted November 19, 1996
ABSTRACT
A protocol for increasing transcription from plasmid expression vectors is
presented. A vector containing chloramphenicol acetyltransferase (CAT) gene was
digested leaving the transcription cassette intact. Heat inactivation of
restriction enzymes followed by ligation of the digestion products yielded
concatemers which migrated as a single band in agarose gel electrophoresis.
Mouse fibroblasts transfected with the concatemers gave a CAT activity that was
14-fold greater than that of cells transfected with a similar mass (equimolar
gene number) of the native plasmid. The effect was independent of promoter
type, restriction enzyme, number of restriction sites and with a noted
exception, cell line.
Plasmid expression vectors are used extensively in molecular biology for a broad
range of purposes. The reliable expression of cloned genes has made the complex
process of eucaryotic transcription amenable to detailed study proving
indispensable for determining the roles played by initiator and regulatory
elements (
1
). In general, strong expression from a vector is a desirable trait and a number
of strategies exist to improve expression levels: site directed mutagenesis of
upstream activation sequences may lead to increased expression (
2
), upstream insertion of tandem repeats of promoters improve expression from
cloned genes (
3
). Expression levels are also a function of promoter type, cell line and
transfection efficiency (
4
). In an earlier study (
5
), we discovered that concatemer plasmids generated by ligating
Nsp
I digests of plasmid expression vectors had increased transcriptional activity.
Here we expand on those results to show that the findings are robust, highly
reproducible, independent of restriction site number, restriction enzyme,
promoter and cell line and may be used as a general method for increasing
transcription levels in plasmid expression vectors up to 14-fold.
Our expression vector (pKC-CAT) is a 3871 base pair (bp) plasmid (Fig.
1
). The transcription cassette occupies 997 bp and comprises a polyomavirus late
promoter, chloramphenicol acetyltransferase (CAT) gene and SV40 polyadenylation
signal. We digested the plasmid with
Nsp
I which yielded two fragments (1930 and 1941 bp respectively), one of which
contained the intact transcription cassette. Heat inactivation of the
restriction enzyme followed by ligation of the fragments at high concentration
of DNA ( >= 1 [mu]g/[mu]l) lead to concatemerization of DNA. Ethidium bromide-stained agarose gel electrophoresis showed the concatemers
migrating as a single broad, slow-moving band indicating sizes well in excess of the largest band (12 kb) in
the l kb size markers from Gibco BRL (Fig.
2
). It is not known whether the products formed were linear or circular. Electron
micrographs made in an earlier study show that the DNA of the concatemers
resembled electron dense `tangles of shoe-laces' instead of the more usual diffuse pattern associated with native
plasmid DNA. Using the method of Chen and Okayama (
8
), which uses a BES based 2* buffer rather than the conventional HEPES based one, calcium phosphate-mediated transfections of 10 [mu]g concatemers into 100 mm plates of 30-50% confluent NIH 3T3 cells were carried out. CAT
activity was assayed 48 h later by a phase extraction method (
6
). CAT activity was 14-fold higher than that achieved using a control (similar mass of the native
plasmid; equimolar with respect to gene number; Fig.
3
). In an earlier study (
5
) we used slot blot analysis of total DNA extracts of calcium phosphate
transfected cells to show that copy number of the gene was the same for cells
transfected with either the concatemer or the native plasmid. This indicated
that enhanced expression was not due to different transfection efficiencies.
Differences in hybridization efficiency of the two forms of DNA were also ruled
out (
5
). We were able to reproduce the effect using concatemers generated from
fragments made with a different restriction enzyme (
Bsa
I; has a unique site outside the transcription cassette), when using a different
promoter (pSV2CAT is a CAT expression vector containing an SV40 promoter and
may be digested at a unique site outside the transcription cassette by the
restriction enzyme
Apa
I) when using a cell line of non-fibroblast origin (Hu H7 is a human hepatoma cell line established from a
hepatocellular carcinoma; ref.
9
; Fig.
3
). When
Nco
I, which has a unique restriction site inside the transcription cassette was
used to generate fragments, the concatemers showed a 50% loss of activity (Fig.
3
). We conclude that even when the restriction site lies in the transcription
cassette, 50% of the linear fragments generated will re-ligate in the right context. The loss of activity was even greater (~95%) when
Eco
RI was used (data not shown). The latter has three restriction sites, one of
which lies in the transcription cassette, making it unlikely for the three
fragments generated to religate into the right context.
4 Sambrook,J. Fritsch,E.F. and Maniatis,T. (1989) In Ford,N. (ed.) Molecular Cloning: A Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
5 Leahy,P. Carmichael,G.G. and Rossomando,E.F. (1996) Bioconjugate Chem., 7, 545-551.
6 Leahy,P., Carmichael,G.G. and Rossomando,E.F. (1995) BioTechniques19, 894-898.MEDLINE Abstract
7 Selden,R.F. (1987) In Ausubel,F.M., Brent,R., Kingston,R.E., Moore,D.D., Seidman,J.G. Smith,J.A. and Struhl,K. (eds) Current Protocols in Molecular Biology. Wiley Interscience, New York. Section 9.
8 Chen,C. and Okayama,H. (1987) Mol. Cell Biol. 2, 1044-1051.
9 Nakabayashi,H., Taketa,K., Miyano,K., Yamane,T. and Sato J. (1982) Cancer Res. 42, 3858-3863.MEDLINE Abstract
10 Franks,R.R., Hough-Evans,B.R., Britten,R.J. and Davidson,E.H. (1988) Development 102, 287-299.
*
To whom correspondence should be addressed at Department of Biochemistry, Case
Western Reserve, University School of Medicine, 10900 Euclid Avenue, Cleveland,
OH 44106-4935, USA. Tel: +1 216 368 3634; Fax: +1 216 368 4544; Email:
pxl24@po.cwru.edu
