Biology Reference
In-Depth Information
virus-polybrene complex to the adipocyte surface and thereby increases the uptake of
the virus into the cells. We have also found that the adherent adipocytes are highly
endurant of any potential stress caused by such centrifugation, as they stay healthy with
intact lipid droplets and responsive to hormones such as insulin and isoproterenol after
the procedure. Therefore, of all methods developed for ectopic gene expression in
adipocytes, centrifugation-mediated enhancement of adenoviral infection demon-
strates considerable promise because it permits a high-efficiency transduction in
mature 3T3-L1 adipocytes known to express receptors for adenovirus at low levels.
The fact that cells can easily reattach to culture plates after electroporation has
made it possible to conduct siRNA knockdown and adenoviral overexpression in
a sequential manner. This is especially useful when the interplay between two dif-
ferent proteins needs to be interrogated. As described in the case study in
Section 2.4
,
we were able to evaluate the impact of CGI-58 or perilipin 1 on ATGL action through
overexpression of ATGL with or without simultaneous knockdown of endogenous
CGI-58 or perilipin 1. Since the studies are conducted in fully differentiated adipo-
cytes, these methods enable the investigators to exclude any confounding effects of
potential developmental or adipogenic defects caused by loss- or gain-of-function
manipulations. Furthermore, though they were specifically developed for studying
hormone-stimulated lipolysis, the techniques should be equally useful for elucidating
protein functions related to many other aspects of adipocyte biology.
Acknowledgment
This work was supported by research grants from the National Institutes of Health
(DK089178) and the American Diabetes Association (1-10-JF-30) to J. L.
References
