Fluorescent Markers For Computer Modeled Antisense Peptide Nucleic Acids Against Simian Virus 40 Large T-Antigen
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Author/Creator ORCID
Date
2000-05
Department
Hood College Biology
Program
Biomedical and Environmental Science
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Abstract
Peptide Nucleic Acids (PNA's) are DNA analogues which
have had the traditional phosphodiester backbone replaced by
an uncharged peptide backbone. PNA's have shown promise as
possible antisense inhibitors of gene expression (Hanvey et
al. 1992). The study of the antisense effects of a PNA is
demonstrable using microinjection to deliver the PNA into a
cell's nucleus along with a gene marker of microinjection
efficiency, beta galaactocidase (Bgal), and target gene
Simian Virus 40 large T antigen (5V40 TAg). Expression of
SV40 TAg in comparison to marker Bgal expression is assayed
by dual immunofluorescence. For this thesis, an antisense
PNA was targeted against SV40 TAg, and a novel reporter
system was devised. The first criterion for the selection of
an antisense PNA against SV40 TAg was that the PNA mismatch
any known human gene by at least two bases. The second
criterion for antisense PNA selection was to find a target
site as near to the SV40 TAg translation start site as
possible. FastA searches of available genetic databases
such as GenBank were used to locate a target site in SV40
TAg mRNA. Computer models of the SV40 TAg mRNA secondary
structure predicted that this sequence, an 18mer, was part
of a stem loop structure thirteen bases downstream of the
start site with ten unpaired bases comprising a loop
structure and the rest of the bases involved in intramolecular
base pairing within the stem. The antisense PNA
selected by these computer searches, AS7, was constructed
and tested in several cultured cell types. The inhibitory
effect achieved by AS7 was nearly 100% with no discernable
antisense side effects. For the detection of antisense
effect, a Green Fluorescent Protein - SV40 TAg plasmid
(GFPT) was constructed which expressed SV40 TAg fused to the
amino end of Green Fluorescent Protein(GFP). GFPT provided
target gene expression with high efficiency. Three markers
of microinjection efficiency were tested. Markers, in
descending order of effectiveness, were BGal, red
fluorescent beads, and blue fluorescent protein (BFP).