Alphavirus-Based Vectors - Viral Vector Approaches in Neurobiology and Brain Diseases - page 98

3.3 In Vitro
Transcription

To achieve high-titer virus stocks, it is essential to generate high
quality in vitro transcribed RNA. It is advisable to prepare fresh
RNA preparations for each electroporation although RNA tran-
scripts can be stored for shorter periods (weeks) at −80 °C. The
procedure can be scaled up by multiplying the volumes for the in
vitro transcription reactions and performing multiple electropora-
tions in parallel. It is important to set up the in vitro transcription
reactions at room temperature as the SP6 buffer contains spermi-
dine, which might lead to precipitation at lower temperatures. Add
the enzyme components last. Set up separate in vitro transcription
reactions for expression and helper vectors in sterile 1.5-mL micro-
centrifuge tubes ( see Note 2 ).

Although manufacturers provide their own in vitro transcription
buffer with their SP6 RNA polymerase, it is recommended to use
the optimized buffer [ 5 ] below.

3.3.1 SFV In Vitro
Transcription Reaction

5 μ L (2.5 μ g) linearized plasmid DNA

5 μ L 10× SP6 buffer

5 μ L 10 mM m 7 G(5 ′ )ppp(5 ′ )G

5 μ L 50 mM DTT

5 μ L rNTP mix (10 mM rATP, 10 mM rCTP, 10 mM rUTP, and

5 mM rGTP)

x μ L RNase-free H 2 O to reach a fi nal volume of 50 μ L

1.5 μ L (50 U/ μ L) RNase Inhibitor

3.5 L) SP6 RNA polymerase

Mix all reaction components in the listed order and spin briefl y
in a microcentrifuge. Incubate 1 h at 37 °C ( see Note 3 ). Load
1-4

μ L (20 U/ μ

L aliquots for RNA quality control on 0.8 % agarose gel.
Continue the incubation for the rest of the samples. Thick bands
without smearing indicate high-quality RNA. RNA from expres-
sion vectors has an approximate (depending on insert size) mobil-
ity of 8 kb (compared to DNA markers), whereas helper RNA runs
faster. Generated RNA molecules can be directly subjected to elec-
troporations or stored for weeks at −80 °C. Frozen RNA samples
should be reevaluated before use. The yields from transcription
reactions should be in the range of 20-50

μ

μ g of RNA.

BHK-21 cells produce high-titer SFV stocks. Alternative host cells
can be considered. The cells should only possess a low passage
number (cultured <3 months) to provide high viability.
Furthermore, cells should be cultured in T175 fl asks to no more
than 80 % confl uency as old and too dense cell cultures generate
signifi cantly lower virus titers ( see Note 4 ). Wash cells once with
PBS and trypsinize with 6 mL trypsin-EDTA per T175 fl ask for
5 min at 37 °C. Resuspend cells well to remove clumps and add
cell culture medium to 25 mL. Centrifugate for 5 min at 800× g

3.4 Electroporation
of RNA