Alphavirus-Based Vectors - Viral Vector Approaches in Neurobiology and Brain Diseases - page 97

stock), 2 mM MgCl (1/500 of stock), and 1 mg/mL X-gal
(1/20 of stock)
46. Moviol 4-88 containing 2.5 % DABCO (1,4-diazobicyclo-
[2.2.2]-octane)
47. Lysis buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 2 mM
EDTA, and 1 % (v/v) Nonidet P-40 (NP40)) (Sigma)
48. Hybond ECL nitrocellulose fi lter (Amersham)
49. TBST (TBS with 0.1 % Tween 20)
50. ECL Chemiluminescence kit (Amersham)
51. Starvation medium (Methionine-free MEM, 2 mM glutamine,
and 20 mM HEPES)
52. Chase medium (E-MEM, 2 mM glutamine, 20 mM HEPES,
and 150 μ g/mL unlabelled methionine)

3 Methods

The subcloning of genes of interest into the multiple cloning sites
(MCS) of the SFV expression vectors are conducted according to
general cloning procedures. The large size of the SFV vectors
makes it favorable to initially clone PCR fragments into other types
of cloning vectors such as the pCR4Blunt-TOPO vector
(Invitrogen) and thereafter subcloning into SFV vectors. Because
the region between the MCS and the linearization sites contains
the RNA replication and polyA + signals the linearization sites ( Spe I,
Sap I, and Nru I) in SFV cannot be used as cloning sites. Likewise,
the unique Xmn I site (at position 9929) in the pSFV1 vector is
inappropriate as the RNA transcript becomes too long to function
properly. Restriction endonuclease digestions and nucleotide
sequencing are applied for the verifi cation of inserts. It is recom-
mended to prepare DNA Midiprep or Maxiprep DNA templates
for in vitro transcription reactions. Initial transcription tests can,
however, be carried out with Miniprep DNA ( see Note 1 ).

3.1 Subcloning
into SFV Vectors

3.2 DNA
Linearization

SFV plasmid vectors are linearized by Spe I, Sap I, or Nru I under
standard restriction digestion conditions in quantities of 5-10 g

plasmid DNA (larger quantities can be stored at −20 °C). Digestions
are confi rmed by agarose gel electrophoresis in comparison to
uncut plasmid and the linearized DNA is purifi ed by phenol/chlo-
roform extraction followed by ethanol precipitation (over night at
−20 °C or 15 min at −80 °C). Centrifugate ethanol precipitates
15 min at 18,000× g at +4 °C and wash with 70 % ethanol. Repeat
centrifugation for 5 min, air dry or lyophilize the DNA pellet and
resuspend in RNase-free H 2 O at a fi nal concentration of 0.5

μ

L.
Alternatively, MicroSpin™ S-200 HR Columns (Amersham) can
be applied according to the manufacturer's instructions.

μ g/ μ