Læknaneminn - 01.10.1996, Blaðsíða 99

Rannsóknarverkefni 4, árs læknanema, útdrættir
Semliki forest virus transfection as a means to
generate antisense RNA in living cells
Anna Marprét Halldórsdóttir1.
Anke Huckriede2, Astrid Heikema2
'LHI, "Laboratory of Physiological Chemistry,
University of Groningen.
Introduction: Antisense RNA molecules complementary
to mRNAs can inhibit (or reduce) expression of cellular genes
in a specific manner. They have been used extrensively to ex-
perimentally control expression of different proteins in
eukaryotic cells and offer a model for future specific gene
therapy. Currently used approaches in applying antisense
RNAs to cells have many drawbacks, including inefficiency
and being only suitable for in vitro experiments.
The aim of this project is to explore whether Semliki for-
est virus mediated transfection is suitable for the generation
of antisens RNA in living cells. The SVF transfection system
is an efficient system for the transient expression of recomb-
inant proteins. It involves cloning of the gene of interest into
a plasmid (pSFVs) containing a DNA copy of a part of the
SFV RNA genome. After transcription of plasmids, contrans-
fection of recombinant RNA and helper RNA, encoding the
structure virus proteins, results in production of recombinant
SFV particles. First the effect of “empty” SFV particles
(encoding viral genes only) on cellular protein synthesis in
general has to be determined. If this proves to be extensively
impaired, SFV will not be useful as an antisense RNA “vector”,
as the aim is to inhibit production of only one specific prot-
ein, with as little effect on the general cell function as possible.
Materials and methods: First the “empty” SFV particles
had to be produced: Linearized and purified pSFVj and
pHelperl plasmids were transcribed in vitro to produce
complementary RNAs, which were checked on gel and ther-
eafter coelectroporated into Baby Hamster Kidney (BHK21)
cells. The cells were grown for 26 hours and cell medium
removed. To obtain purified viral particles for character-
ization, part of the medium was ultracentrifugated on a
sucrose gradient. Viral proteins were separated using SDS-
PAGE and detected on Western blots using antibodies spe-
cific to the viral structural protein E1. BHK 21 cells were in-
fected with the “empty” SFV particles by applying 500 and
lOOOpl of medium containing the particles to cell cultures.
Six hours after infection effects on protein synthesis were det-
ermined by a pulse chase experiment. Noninfected cells and
cells infected with SFV-DHFR recombinant served as con-
trols. After incubation with radioactive 35S-Met for 20 min,
cells were washed, lysed and radiactivity determined by scin-
tillation counting and fluorography.
Results: Production of “empty” SFV particles was success-
ful. Western blots showed clear bands of the E1 protein that
were on the same height as those of purified original SFV.
Comparison with another recombinant SFV stock showed
that the amount of virus particles obtained was adequate for
infection purposes. Data from the pulse-chase are not yet
complete. Scintillation counts gave bizarre results, the prot-
ein synthesis being increased in cells infected with 1 OOOpl but
lowered in cells infected with 500pl of empty virus particles.
Validity of data is uncertain. Preliminary fluorography
showed no obvious change in protein production in cells in-
fected with 1000 or 500 U1 of empty SFV particles, com-
pared to uninfected cells.
Discussion: Available results are too preliminary to draw
definite conclusions about the effects of “empty” SFV part-
icles on host cell protein synthesis, however the results of the
initial experiment are promising in the way that protein synt-
hesis seems not to be inhibited. More pulse-chase ex-
periments should though be conducted, preferably using
several time-points postinfection rather than one. If this
empty particle proves to have no significant effects on cell
protein production, SFV will be tried out as an antisense
vector. The other outcome - definite inhibition of protein
synthesis - would still be interesting, as it suggests that synt-
hesis of virus structural proteins is not the only reason for
decreased protein production in SFV infected cells.
Tap á arfblendni á litningi 7q í brjóstakrabba-
meini; samband við klínískar breytur
Árni Kjalar Kristiánsson
Guðný Eiríksdóttir 2, Sigurður Ingvarsson 2.
'LHÍ, "Frumulíffræðideild Lsp.
Inngangur: Brjóstakrabbamein er algengasta krabbamein-
ið meðal íslenskra kvenna og veldur flestum dauðsföllum sem
rekja má til krabbameina. Breytingar í erfðamengi
brjóstakrabbameinsfruma eru algengar og taldar eiga þátt í
myndun og þróun krabbbameins. Þessar breytingar má
flokka í þrennt. I fyrsta lági punktstökkbreytingar í æxlisgen-
um og æxlisbæligenum sém eru fremur sjaldgæfar í brjósta-
krabbameinum. I öðru lagi magnanir á æxlisgenum og loks
úrfellingar á litnin^asvæðum sem bera æxlisbæligen, en það
eru algengustu breytingarnar í brjóstakrabbameinum. Afurð-
ir æxlisbæligens gegna vaxtarhindrandi hlutverki. Til að æxlis-
bæligen verði óstarfhæft þurfa yfirleitt báðar samsætur (allele)
þess að vera óvirkar. Önnur samsætan skemmist oft með
stökkbreytingu en hin vegna úrfellingar á Iitningabút sem
hefur að geyma samsætuna, þannig að einstaldingur sem var
arfblendinn fyrir samsætuna er orðinn arfhreinn, þ.e. hefur
tapað arfblendni. Stökkbreytingin á fyrri samsætunni er í
raun víkjandi breyting og getur erfst milli kynslóða sem kím-
línubreyting og gert viðkomandi einstakling útsettari fyrir
krabbamein síðar á ævinni.
Litningarannsóknir og sameindaerfðafræðirannsóknir hafa
sýnt úrfellingar á 7q í ýmsum krabbameinum, þ.á.m.
brjóstakrabbameinum.
LÆKNANEMINN
89
2. tbl. 1996, 49. árg.