Viral Vectors to Study Synaptic Function - Viral Vector Approaches in Neurobiology and Brain Diseases - page 118

Linearization of DNA
and RNA Preparation

(a) Prepare clean recombinant vector DNA and helper DNA plas-
mids using a standard DNA purifi cation protocol. An RNase-
free purifi ed product is recommended to avoid degradation of
your transcripts after in vitro transcription.
(b) Linearize 5-10 g of recombinant vector DNA and helper

DNA plasmids. Choose an enzyme that has a unique site (XhoI
or PacI or NotI) after the PolyA signal and does not cut in the
insert (see Fig. 1 ). The helper DNA should be linearized with
XhoI. Each in vitro transcription will require 1

μ

μ g of linearized

template.
(c) Purify the linearized plasmids with a DNA purifi cation kit.
Resuspend the linearized plasmids in RNase-free TE or water.
Optional: check an aliquot of the linearized plasmids on an
agarose gel to check the quality and quantity.
(d) Perform an in vitro transcription reaction for each plasmid
using commercially available kits (e.g., mMESSAGE mMA-
CHINE ® SP6 Kit).

g of
linearized template. At this point, the RNA can be aliquoted in
10

A typical reaction should yield 10-20 μ g of RNA from 1 μ

L samples and frozen at −80 °C for later use. Optional: check
the quality and/or quantity of the RNA by gel electrophoresis
and/or by standard quantifi cation measurements.

μ

The transfection procedure is described here for production of one
virus. Perform the exact same steps in parallel for each virus to be
produced.

Transfection of BHK Cells
for Virus Production

(a) Prepare a 70 % confl uent 150 mm 2 plate of BHK cells per
virus. It is best to split cells the day before transfection. BHK
cells grow best in MEM medium supplemented with 5 % FBS

and (optional) antibiotics (e.g., streptomycin + penicillin).

α

(b) Wash plate with PBS with cations.
(c) Collect cells by trypsinization.
(d) After centrifugation (2 min at 1,000 rpm), discard superna-
tant and resuspend cells in 10 mL of

α MEM without FBS

serum.
(e) Centrifuge again, discard supernatant and resuspend cells in
10 mL of RNase-free PBS without cations.
(f) Determine number of cells using a counting chamber.
(g) Centrifuge cells, discard supernatant and resuspend cells in
RNase-free PBS without cations at a concentration of
10 7 cells/mL. BHK cells prepared for electroporation must
be used immediately and cannot be stored.
(h) During centrifugation in step (g), place a 0.4-cm electropora-
tion cuvette on ice and add 11 mL of α MEM medium + FBS

to a sterile fi ltered top 150-mm 2 fl asks.