Biology Reference
In-Depth Information
that every cell in the culture is infected. On the contrary, low
MOI is used when multiple cycles of infection are required.
Furthermore, the MOI to be used depends on the gene to be
transduced (e.g., gene product can be toxic for the NPCs) and
should be assessed empirically for any single application.
11. Depending on the MOI used to transduce the cells, on the genes
to be delivered, and its biological effects, NPCs can show altered
phenotype and features. If NPCs appear suffering (i.e., attached
to the culture dish or swollen) it might be helpful to wash them
12 h (instead of 24) after the infection to clear the culture
medium of debris and toxic substances. Again, it is important to
replate NPCs at high density to favor their recovery.
12. Efficiency of infection can be evaluated in several manners
depending on the gene(s) encoded in the transfer vector. For
vectors carrying fluorescent reporters, the easiest and fastest
analysis will be carried out by flow cytometry. On the other
hand, an accurate evaluation of the efficiency of infection for
those vectors, which do not carry any reporter, is the evalua-
tion of the average number of integrated proviral DNA copies
by real-time PCR. Similarly, different assays can be used in
order to evaluate the expression and functional effect(s) of the
delivery of a gene of interest. These include flow cytometry,
Western Blot, immunofluorescence, real-time PCR, enzyme-
linked immunosorbent assay (ELISA), etc. Moreover, it is
advisable to verify if the newly generated NPC lines expressing
one (or more) gene of interest still retain their stemness. This
can be easily done by performing growth assays (e.g., Growth
curve), clonogenic assays (e.g., neurosphere formation assay),
and differentiation assay upon growth factor withdrawal.
13. During administration the vein should blanch and no material
or swelling should be detectable at the injection site. A suc-
cessful injection is obvious to the user, based on lack of resis-
tance, as the plunger is depressed. Preferably the needle should
be inserted into a lateral vein midway down the tail, permit-
ting additional attempts proximally if the initial attempt is
unsuccessful. In most cases an injection volume of 0.2-0.5 ml
can be safely given to an adult mouse. Most users try to limit
the injection to 0.25 ml, a standard veterinary recommenda-
tion, but for cell injections, overconcentration may lead to
embolism in lung capillaries and death of the recipient. The
tendency to clump is cell line dependent.
14. Intraperitoneal injection of ketamine and xylazine (100 mg/
kg and 10 mg/kg, respectively) can be used for young animals
from postnatal day 7 and for adults as well. Volatile anesthesia
with isoflurane is a convenient method for adult animals, guar-
anteeing rapid recovery. However, contrary to ketamine and
xylazine, it doesn't have intrinsic analgesic effects. Analgesics
Search WWH ::
Custom Search