Chemistry Reference
In-Depth Information
purified by Qiagen Mega plasmid purification kit. For transfection by DDMC/DNA,
HEK293 cells (15
10
4
cells) were seeded onto 35-mm culture dishes and
incubated at 37
C in a humidified atmosphere of 5 % CO
2
. In a sterile tube,
10
L of the autoclaved
DDMC having a concentration of 10 mg/mL was added to the DNA solution and
mixed by vortexing briefly. The growth medium (Dulbecco's modified medium
with 10 % fetal calf serum) was removed from the cells to be transfected. The cells
were washed twice with 1
μ
g of DNA was dissolved in 270
μ
Lof1
PBS. Then, 14
μ
PBS. DDMC/DNA complex in solution was added to
cover the cells. The dish was careened slowly several times to ensure complete
coverage of the cells and incubated at 37
C for 30 min. The dish was slowly
careened several times during the incubation and then 1 mL of growth medium was
added and the mixture incubated at 37
C for 48 h. After the incubation, transfection
activity was determined using the X-gal staining method. Following the transfec-
tion protocol, transfection of HEK293 cells with samples of DDMC1 and DDMC2
was carried out using this plasmid DNA. As shown in Fig.
6.29
, a higher value of
five times or more was confirmed for samples of DDMC1 and DDMC2 than for the
starting DEAE-dextran hydrochloride. From the results, the transfection efficiency
and the reaction rate of formation of the complex should increase when using
DDMC hydrochloride instead of DEAE-dextran hydrochloride.
6.4.5.1 Charge Ratio of DNA/DDMC
Definition of charge ratio.
The ratio of phosphorus content to nitrogen content
(P/N) is defined as the charge ratio. With the complex formation reaction between
DDMC (N content 1.4 %) and DNA (P content 9.3 %), the compound is formed by
ionic bonding (polyion complex), and thus the constituent ratio is expressed as the
weight ratio along with the charge ratio as the primary constituent element.
=
ð
Þ¼ðy
:
Þ=ðx
:
Þ
P
N
charge ratio
0
093
14
0
014
31
(6.36)
where DNA/DDMC
¼ y
/
x
(weight ratio); P content is 9.3 %; N content is 1.4 %; P
atomic weight is 14; N atomic weight is 31;
y
is the amount of DNA and
x
is the
amount of DDMC.
P/N ratio.
When considering transfection efficiency, the charge ratio (P/N) of
each sample is an important value as well as the concentration. It is thus necessary
to make the P/N values equivalent when comparing RLU (relative luciferase
expression) values for DEAE-dextran and DDMC. When investigating related
P/N ratios, for example, the percentage of N in DEAE-dextran was 3.3 % and the
percentage of P in the DNA was about 9.3 %. The P/N values shown in Table
6.8
were thus obtained.
With regard to dependence of the amount of transferred DNA on P/N ratio
shown in Table
6.8
, it would appear that a comparison can be made of the RLU
values with DEAE-dextran (graft ratio 0 %) and DDMC (graft ratio 130 %) at the
respective sample P/N values. In other words, a P/N value of 0.037 is given for
DDMC (graft ratio 130 %) at a concentration of 28.6 mg/mL based on the fact that
