recommended that the optimized SP6 RNA polymerase buffer (see
Subheading 2.1) is used instead of available commercial in vitro
transcription buffers (see Note 2). Add enzyme components last
because they are the least stable reactants in the reaction.
In Vitro Transcription Reaction
5 μL (2.5 μg) linearized plasmid DNA.
5 μL 10Â SP6 buffer.
5 μL 10 mM m
7 G(5
0 )ppp(5
0 )G.
5 μL 50 mM DTT.
5 μL rNTP mix.
x μL RNase-free H 2 O to reach a final volume of 50 μL.
1.5 μL (50 U/μL) RNase inhibitor.
3.5 μL (20 U/μL) SP6 RNA polymerase.
1. All reaction components are mixed and incubated for 1 h at
37
C. However, in case of large inserts, yields can be improved
by prolonging the incubation time (see Note 3).
2. The quality of in vitro–transcribed RNA is assessed by loading
1–4 μL aliquots on 0.8% agarose gels. High-quality RNA is
visible as thick bands without smearing along the axis of migration with an approximate mobility of 8 kb (compared to DNA
markers) from the expression vector and a slightly faster mobility of helper RNA. Each in vitro transcription reaction is anticipated to generate 20–50 μg of RNA. The RNA is quantified by
comparison of the intensity of RNA bands to DNA markers or
by spectrophotometric measurements.
3. In vitro–transcribed RNA is directly applied to electroporation
or lipid-mediated transfection. In case RNA transcripts are
stored at À80
C, the quality of RNA should be reevaluated
by agarose gel electrophoresis before use.
3.4 Electroporation
of RNA
Generally, the use of BHK-21 cells is preferred for alphavirus production as they are known to produce high-titer virus stocks
[2]. Alternative host cells can be considered, but the production
process needs to be optimized for each cell line employed. The
following conditions have been found optimal for obtaining hightiter SFV stocks (e.g., 1 Â 10
9 infectious particles/mL) in BHK-21
cells.
1. Cells are cultured under 85–95% humidity and 5% CO 2 with a
low passage number in T175 flasks to no more than 80%
confluency, washed once with PBS, and treated with 6 mL
trypsin–EDTA per T175 flask for 5 min at 37
C to detach
the cells.
70
Kenneth Lundstrom
Subheading 2.1) is used instead of available commercial in vitro
transcription buffers (see Note 2). Add enzyme components last
because they are the least stable reactants in the reaction.
In Vitro Transcription Reaction
5 μL (2.5 μg) linearized plasmid DNA.
5 μL 10Â SP6 buffer.
5 μL 10 mM m
7 G(5
0 )ppp(5
0 )G.
5 μL 50 mM DTT.
5 μL rNTP mix.
x μL RNase-free H 2 O to reach a final volume of 50 μL.
1.5 μL (50 U/μL) RNase inhibitor.
3.5 μL (20 U/μL) SP6 RNA polymerase.
1. All reaction components are mixed and incubated for 1 h at
37
C. However, in case of large inserts, yields can be improved
by prolonging the incubation time (see Note 3).
2. The quality of in vitro–transcribed RNA is assessed by loading
1–4 μL aliquots on 0.8% agarose gels. High-quality RNA is
visible as thick bands without smearing along the axis of migration with an approximate mobility of 8 kb (compared to DNA
markers) from the expression vector and a slightly faster mobility of helper RNA. Each in vitro transcription reaction is anticipated to generate 20–50 μg of RNA. The RNA is quantified by
comparison of the intensity of RNA bands to DNA markers or
by spectrophotometric measurements.
3. In vitro–transcribed RNA is directly applied to electroporation
or lipid-mediated transfection. In case RNA transcripts are
stored at À80
C, the quality of RNA should be reevaluated
by agarose gel electrophoresis before use.
3.4 Electroporation
of RNA
Generally, the use of BHK-21 cells is preferred for alphavirus production as they are known to produce high-titer virus stocks
[2]. Alternative host cells can be considered, but the production
process needs to be optimized for each cell line employed. The
following conditions have been found optimal for obtaining hightiter SFV stocks (e.g., 1 Â 10
9 infectious particles/mL) in BHK-21
cells.
1. Cells are cultured under 85–95% humidity and 5% CO 2 with a
low passage number in T175 flasks to no more than 80%
confluency, washed once with PBS, and treated with 6 mL
trypsin–EDTA per T175 flask for 5 min at 37
C to detach
the cells.
70
Kenneth Lundstrom