amaxa news #10 - Lonza AG

› FAQ
FAQs on Nucleofection ® and
Preservation of Functionality
› Are the rat and mouse hepatocytes still
functional after nucleofection ® ?
›› Yes, transfected mouse hepatocytes show
albumin synthesis rates comparable to those
of untransfected cells. Moreover, the morphology
and polarization of rat hepatocytes is not
affected by nucleofection ® .
› Are there any effects on the differentiation
of neural stem cells following nucleofection ® ?
›› The ability of transfected rat or mouse neural
stem cells to be subsequently differentiated
into a variety of cell types is well documented
in scientific literature, for example:
› Nucleofection ® of neural stem cells (NSC)
does not affect their functionality or impinge
on their ability to be subsequently differentiated
into oligodendrocytes [Copray et al.
(2006) Stem Cells 24(4): 1001-1010].
› Have you analyzed whether nucleofection ®
influences differentiation in mouse ES cells?
›› During the optimization process of a
Nucleofector ® Protocol, great care is taken
about preservation of functionality of the
cells post transfection. Internal experiments
showed that nucleofection ® does not alter the
differentiation ability of mouse ES. This was
investigated by analyzing the expression level
of the transcription factor GATA-6. Results
have also shown that the differentiation process
itself is not triggered by nucleofection ® .
This is well documented in literature showing
unaltered differentiation potential of mouse
ES cells, e.g. (Lakshmipathy et al., Stem Cells
22:531-543).
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› Have you tested whether transfected macrophages
maintain functionality?
›› Yes, we analyzed the secretion of TNF-a triggered by stimulation
of the cells with LPS 24 hours post nucleofection ® . In comparison
to the untransfected control, the functionality is 70%.
› Is it possible to stimulate transfected T cells?
›› Yes, transfected resting T cells can be stimulated post
nucleofection ® . It is important to wait at least 4 hours before
stimulation (e.g., incubation in Human T Cell Medium provided
by amaxa). Stimulating immediately after nucleofection ® may
lead to increased cell mortality and poor activation of cells.
For optimal stimulation of the transfected cells, we recommend
coating 96-well plates for stimulation with a solution of
anti-CD3 antibody [OKt 3; eBioscience, Cat. No. 14-0037-82]
and anti-CD28 antibody [15E8; Research Diagnostics Inc.,
Cat. No. RD1-111650clb]. For stimulated cells, maintenance of the
stimulated state was analyzed by expression of CD25 as well as
the secretion of interferon gamma.
› Is it possible to transfect mRNA of tumor cells into
antigen-presenting cells like B cells or dendritic cells
with the Nucleofector ® Technology?
›› Yes. Recently Coughlin and co-workers showed that RNA
transfected CD40-activated B cells or DCs induce functional
T cell responses against viral and tumor antigen targets
(Coughlin et al. (2004) Blood 103:2046-2054).
In short, the authors found:
› mRNA uptake in B cells and dendritic cells up to 80%.
› CD40-activated B cells or dendritic cells induce functional
T cell responses against viral and tumor antigen targets.
› mRNA transfection is less toxic and gave higher efficiencies.
amaxa web information www.amaxa.com/faq