1. Recombinant SFV plasmids are linearized by SpeI or NruI
under standard restriction digestion conditions.
2. Complete digestions are confirmed by agarose gel electrophoresis and linearized DNA constructs are purified by phenol/
chloroform extraction followed by precipitation with 0.3 M
sodium acetate (final) and 2.5Â volume of 95% ethanol (overnight at À20
C or 15 min at À80
C).
3. The ethanol precipitates are centrifugated for 15 min at
18,000 Â g at +4
C and washed with 70% ethanol. DNA
pellets are air-dried or lyophilized after repeated centrifugation
for 5 min and resuspended in RNase-free H 2 O at a final concentration of 0.5 μg/μL (measured in a spectrophotometer).
Alternatively, MicroSpin™ S-200 HR Column purification can
be used for DNA purification.
3.3 In Vitro
Transcription
It is highly recommended that each batch of in vitro–transcribed
RNA is prepared fresh for immunizations or electroporations,
although RNA transcripts can be stored for shorter periods (e.g.,
weeks) at À80
C. It is most important to set up the in vitro
transcription reactions at room temperature as the spermidine in
the SP6 buffer might precipitate at lower temperatures. It is
ReplicaƟon-deficient parƟcles
ReplicaƟon-proficient parƟcles
Layered DNA-RNA vectors
nsP1 nsP2 nsP3 nsP4
Foreign
pA
C
E3
E2
E1
pA
nsP1 nsP2 nsP3 nsP4
Foreign
nsP1 nsP2 nsP3 nsP4
Foreign
pA
C
E3
E2
E1
pA
C
E3
E2
E1
nsP1 nsP2 nsP3 nsP4
Foreign
pA
Fig. 2 Schematic illustration of alphavirus vectors. The application of different alphavirus vectors for
expression studies: Replication-deficient particles are generated by cotransfection of expression vector
and helper vector RNA into BHK-21 cells. Replication-proficient particles are produced by transfection of a
full-length RNA genome including the foreign gene of interest introduced downstream of the nonstructural
replicase genes (nsP1–4) or the structural genes (C-E3-E2-E1). DNA-RNA layered vectors are used for direct
plasmid DNA transfections. SP6 RNA polymerase promoter illustrated as light gray triangle, subgenomic 26S
promoter as dark gray triangle, and CMV promoter as white triangle
Alphavirus-Based Antigen Preparation
69
under standard restriction digestion conditions.
2. Complete digestions are confirmed by agarose gel electrophoresis and linearized DNA constructs are purified by phenol/
chloroform extraction followed by precipitation with 0.3 M
sodium acetate (final) and 2.5Â volume of 95% ethanol (overnight at À20
C or 15 min at À80
C).
3. The ethanol precipitates are centrifugated for 15 min at
18,000 Â g at +4
C and washed with 70% ethanol. DNA
pellets are air-dried or lyophilized after repeated centrifugation
for 5 min and resuspended in RNase-free H 2 O at a final concentration of 0.5 μg/μL (measured in a spectrophotometer).
Alternatively, MicroSpin™ S-200 HR Column purification can
be used for DNA purification.
3.3 In Vitro
Transcription
It is highly recommended that each batch of in vitro–transcribed
RNA is prepared fresh for immunizations or electroporations,
although RNA transcripts can be stored for shorter periods (e.g.,
weeks) at À80
C. It is most important to set up the in vitro
transcription reactions at room temperature as the spermidine in
the SP6 buffer might precipitate at lower temperatures. It is
ReplicaƟon-deficient parƟcles
ReplicaƟon-proficient parƟcles
Layered DNA-RNA vectors
nsP1 nsP2 nsP3 nsP4
Foreign
pA
C
E3
E2
E1
pA
nsP1 nsP2 nsP3 nsP4
Foreign
nsP1 nsP2 nsP3 nsP4
Foreign
pA
C
E3
E2
E1
pA
C
E3
E2
E1
nsP1 nsP2 nsP3 nsP4
Foreign
pA
Fig. 2 Schematic illustration of alphavirus vectors. The application of different alphavirus vectors for
expression studies: Replication-deficient particles are generated by cotransfection of expression vector
and helper vector RNA into BHK-21 cells. Replication-proficient particles are produced by transfection of a
full-length RNA genome including the foreign gene of interest introduced downstream of the nonstructural
replicase genes (nsP1–4) or the structural genes (C-E3-E2-E1). DNA-RNA layered vectors are used for direct
plasmid DNA transfections. SP6 RNA polymerase promoter illustrated as light gray triangle, subgenomic 26S
promoter as dark gray triangle, and CMV promoter as white triangle
Alphavirus-Based Antigen Preparation
69