2. COS7 cells are cultured in DMEM (Dulbecco’s Modified
Eagles Medium) supplemented with 5% fetal calf serum,
5 mM glutamine and 0.1% penicillin/streptomycin.
2.4 Alphavirus
Plasmid Vectors
For the three most commonly used alphaviruses, SFV, SIN and
VEE, similar expression and helper vectors have been engineered
and most methods are the same. However, the focus here is on SFV
only and more detailed information on SIN can be found elsewhere
[27]. The following vectors were applied for generation of replicon
RNA and recombinant viral particles:
1. pSFV1 (basic vector, minimal cloning sites: BamHI, SmaI) [2].
2. pSFV2gen (multilinker cloning sites: ApaI, BamHI, XhoI,
SpeI, SmaI) [28].
3. pSFV-Helper2 (second generation helper vector) [29].
4. pSFV4 (full-length vector) [30].
5. VA7(74) (full-length vector from avirulent strain) [31].
6. pSCA1 (layered DNA-RNA vector with CMV promoter) [32].
7. pSCA-Helper (layered DNA-RNA helper vector with CMV
promoter) [33].
8. pCMV-SFV4 (full length layered DNA-RNA vector) [34].
For the generation of replication-deficient recombinant SFV
particles, either pSFV1 [4] or pSFV2gen (also called pSFV4.2) [28]
vectors are used together with the pSFV-Helper2 [29] vector
(Fig. 2). In contrast, pSFV4 [30] or VA7(74) [31] vectors are
applied for the production of full-length replication-proficient particles. Layered DNA-RNA vectors [33] can be used directly for
transfection of host cells for recombinant proteins or alternatively
together with the layered DNA-RNA helper vector or as a fulllength layered DNA-RNA vector [34] for production of
replication-deficient and proficient particles, respectively.
3 Methods
3.1 Subcloning into
SFV Vectors
Genes of interest can be introduced into the cloning sites of the
chosen SFV expression vector and restriction endonuclease digestions and nucleotide sequencing can be used for verification of
inserts. Preparation of high-purity DNA (Midiprep or Maxiprep
DNA) is highly recommended to provide the best possible quality
and yields of in vitro–transcribed RNA for recombinant viral particle production and/or immunization (see Note 1).
3.2 DNA
Linearization
Efficient in vitro transcription requires complete linearization of
plasmid DNA. The pSFV1 and pSFV-Helper2 vectors are linearized
by SpeI and pSFV2gen by NruI.
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Kenneth Lundstrom
Eagles Medium) supplemented with 5% fetal calf serum,
5 mM glutamine and 0.1% penicillin/streptomycin.
2.4 Alphavirus
Plasmid Vectors
For the three most commonly used alphaviruses, SFV, SIN and
VEE, similar expression and helper vectors have been engineered
and most methods are the same. However, the focus here is on SFV
only and more detailed information on SIN can be found elsewhere
[27]. The following vectors were applied for generation of replicon
RNA and recombinant viral particles:
1. pSFV1 (basic vector, minimal cloning sites: BamHI, SmaI) [2].
2. pSFV2gen (multilinker cloning sites: ApaI, BamHI, XhoI,
SpeI, SmaI) [28].
3. pSFV-Helper2 (second generation helper vector) [29].
4. pSFV4 (full-length vector) [30].
5. VA7(74) (full-length vector from avirulent strain) [31].
6. pSCA1 (layered DNA-RNA vector with CMV promoter) [32].
7. pSCA-Helper (layered DNA-RNA helper vector with CMV
promoter) [33].
8. pCMV-SFV4 (full length layered DNA-RNA vector) [34].
For the generation of replication-deficient recombinant SFV
particles, either pSFV1 [4] or pSFV2gen (also called pSFV4.2) [28]
vectors are used together with the pSFV-Helper2 [29] vector
(Fig. 2). In contrast, pSFV4 [30] or VA7(74) [31] vectors are
applied for the production of full-length replication-proficient particles. Layered DNA-RNA vectors [33] can be used directly for
transfection of host cells for recombinant proteins or alternatively
together with the layered DNA-RNA helper vector or as a fulllength layered DNA-RNA vector [34] for production of
replication-deficient and proficient particles, respectively.
3 Methods
3.1 Subcloning into
SFV Vectors
Genes of interest can be introduced into the cloning sites of the
chosen SFV expression vector and restriction endonuclease digestions and nucleotide sequencing can be used for verification of
inserts. Preparation of high-purity DNA (Midiprep or Maxiprep
DNA) is highly recommended to provide the best possible quality
and yields of in vitro–transcribed RNA for recombinant viral particle production and/or immunization (see Note 1).
3.2 DNA
Linearization
Efficient in vitro transcription requires complete linearization of
plasmid DNA. The pSFV1 and pSFV-Helper2 vectors are linearized
by SpeI and pSFV2gen by NruI.
68
Kenneth Lundstrom