Protocol 
RAC Application 
Kenneth L. Brigham, M.D. 
Research Design and Methods 
Plasmid description and production of plasmid DNA 
The pCMV^ vector (23) was selected for the expression constructs because it represents 
a "state of the art" expression vector. In particular, this vector provides a stable mRNA through 
the use of human growth hormone termination and poly-adenylation signals. This vector also 
provides sequences from the alfalfa Mosaic Virus-4 which act as a translational enhancer by 
decreasing the requirement for initiation factors in protein synthesis. In addition, this 
construct also contains a polylinker region and the promoter-regulatory region of the human 
cytomegalovirus major immediate early gene. 
The PCMV 4 -AAT plasmid was constructed by linker-primer polymerase chain reaction 
(LP-PCR). The coding sequence of PGH synthase was inserted into the PCMV 4 expression 
vector. This method consists of synthesizing two oligonucleotide primers (20-30 nucleotides in 
length). One oligonucleotide is homologous to the 5’ untranslated region immediately upstream 
(5’) of the initiation codon and the second oligonucleotide is complementary to the 3’ 
untranslated region immediately downstream (3’) of the stop transcription codon. Both 
oligonucleotides have a one or two base substitution which creates a unique restriction enzyme 
site in the untranslated regions of the amplified gene. The 5’ and 3’ oligonucleotides were 
designed such that the created restriction enzyme site is approximately 8 nucleotides 
downstream from the 5’ end of the oligonucleotide. Both of these requirements are critical, the 
former to insure a restriction enzyme site w'hich is recognizable and cleavable and the latter 
to insure that the reading frame of the gene is not altered. 
The reading frame of the gene of interest was amplified using Vent DNA polymerase, 
100 ng of target DNA, a programmable temperature cycler, and standard reaction conditions 
(denaturing at 93. 5”, annealing at 56° and extension at 75°). Vent DNA polymerase was used 
because it has a 3’ to 5’ proofreading activity in addition to enhanced stability at high 
temperature and a highly specific and processivc 5’ to 3’ DNA polymerase activity. After PCR 
amplification, the unique restriction sites were cleaved with the appropriate restriction 
enzymes, the amplified gene was separated from the small fragments released by the action of 
the restriction enzymes and from unincorporated primers and nucleotides by gel filtration 
through a S-400 spin column. The amplified genes which now had cloning sites on each end 
were ligated into PCMV 4 which had been previously cleaved with the same restriction enzymes 
which were utilized to prepare the cloning sites on the amplified gene. 
After ligation, the PCMV 4 -AAT construct was transfected into fresh competent bacteria 
(£. coli NM522). The competent bacteria were prepared by standard methods (24). After 
transfection the bacteria were plated out on plates containing carbenicillin, an ampicillin 
analog which provides selection pressure for bacteria containing the PCMV 4 construct. The 
plasmid carries the gene for ampicillin resistance. After the bacteria which harbor the plasmid 
are have grown into distinct colonies, several of the colonies are grown up as individual 5 ml 
liquid cultures. An aliquot of the liquid cultures is stored and the rest is processed as a "mini" 
prep. We then confirm that the isolated plasmid contains the inserted piece of DNA by 
performing both a dot blot analysis and by releasing the inserted piece of DNA by performing 
a restriction enzyme digest. The restriction enzyme digest should yield both the linearized 
plasmid and the original piece of DNA. 
Once a colony has been isolated which contains the piece of cloned DNA, a large scale 
plasmid preparation is grown. The plasmid is purified using the commercial Giga-prep column 
method (Quiagen). Finally, the isolated plasmid is precipitated with ethanol and resuspended 
in sterile water. Samples are stored at -20 '"C in 1 ml aliquots. 
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Recombinant DNA Research, Volume 19 
