Biology Reference
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their effi ciency repeatedly during this procedure. If these sites are
present in your insert of interest, alternative unique sites in the
multiple cloning site (MCS, Fig. 1a ) can be selected and intro-
duced using a similar primer design.
(a) Amplify cDNA of protein-of-interest using the custom-made
primers described above. I recommend the use of Pfu poly-
merase or another high-fi delity DNA polymerase for PCR
amplifi cation to minimize mutagenesis.
(b) Digest both the sindbis virus vector and the PCR product with
XbaI and SphI. Optional: the vector can be 5
dephosphory-
lated using an alkaline phosphatase to prevent self-religation.
(c) Optional: Gel purify or column purify the linearized vector
and PCR product to obtain cleaner material for ligation.
(d) Ligate PCR insert to vector using a T4 ligation kit. A useful
control for the cloning procedure is the preparation of a liga-
tion reaction with the vector alone.
(e) Transform the ligations by electroporation or chemical trans-
formation into E. coli . The number of colonies on the vector
plus insert plate should be several fold higher (minimum of
×2) than on the vector alone plate, suggesting successful
cloning.
(f) Check for correct insertion by restriction digest on mini-
prep DNA (e.g., using a XbaI/SphI restriction digest) or PCR
on single colony (e.g., using the custom-designed primers),
both of which should display a DNA band of the size of the
insert.
(g) Sequence clone of interest to check for eventual unwanted
mutations. The pSINREP5 forward primer is provided in
Fig. 1b . Additional primers within the insert may be needed
for sequencing the entire insert.
During this procedure, we will generate recombinant RNA and
helper RNA for transfection in baby hamster kidney (BHK) cells to
produce infectious viral particles. We will use purifi ed, linearized
pSINRep(nsP2S 726 ) containing our gene of interest (cloned using
the procedure in Sect. 3.1.1 ) and helper DH-BB(tRNA/TE12)
DNA to produce recombinant RNA with an in vitro transcription
kit. The recombinant RNA produced must then be capped and
have a polyA tail, so it will be treated as a messenger RNA when
transfected into the BHK cells. The viral particles produced by the
BHK cells will then be harvested and concentrated prior to in vivo
injection. The end of this protocol needs to be performed in a bio-
safety level 2 tissue culture room using all the safety procedures
designed to work with viruses as indicated below.
3.1.2 Production
and Concentration
of Sindbis Viruses
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