Biomedical Engineering Reference
In-Depth Information
of the HEK 293 cell line reached 100% when the passage exceeded 65, whereas using
low-passage (
<
52) HEK 293 cell line no tumor could be induced under the same con-
dition. Thus, more attention should be paid to the passage level (
<
50) of the HEK 293
cell line.
•
Appropriate pH of 2
HBS
: The optimum pH range for high transfection efficiency is
extremely narrow (between 7.05 and 7.12). The pH meter used to measure the pH of 2
×
×
HBS should be calibrated several times until very accurate. The pH of 2
×
HBS could
20
◦
Cinsmall
aliquots. The solution should be made fresh if transfection has not worked well.
change during thawing and storage. The solution should be stored at
−
•
Precipitates of Ca
3
(PO4)
2
-DNA complexes
: Highly effective precipitates for transfec-
tion purposes can be generated only in a very narrow range of physico-chemical conditions
that control the initiation and growth of the Ca
3
(PO
4
)
2
-DNA complexes. The pH value
and concentrations of phosphate (HBS solution) and calcium (CaCl
2
solution) are the
main factors influencing the characteristics of the Ca
3
(PO
4
)
2
-DNA complexes. It is nec-
essary to check Ca
3
(PO
4
)
2
precipitates when you prepare new 2.5 M CaCl
2
and 2
HBS.
Also, the reaction time of the precipitate complex is very important. Theoretically, the
standing time can be optimized for each set of parameters. It has been found in most cases
that almost all of the soluble DNA in the reaction mix can be bound into an insoluble
complex with Ca
3
(PO
4
)
2
in 1-3min. Extending the reaction time to more than 5min
results in reducing the level of expression.
×
•
Discontinuous iodixanol gradients
: It is important the first iodixanol step (15%) contains
1 M NaCl to destabilize ionic interactions between macromolecules. Without 1 M NaCl in
15% iodixanol the gradients would distribute the vector along the whole length of the
gradient, making separation of virus unsuccessful. High salt needs to be excluded from
the rest of the gradient in order to band the virus under iso-osmotic conditions, allowing
direct loading in subsequent chromatographic steps.
References
1. Smithies, O. (2005) Many little things: one geneticist's view of complex diseases.
Nature Reviews
Genetics
,
6
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2. Gu, H., Marth, J.D., Orban, P.C.
et al
. (1994) Deletion of a DNA polymerase beta gene segment
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et al
. (2007) Local production of angiotensin II in
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Journal of Clinical Investigation
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, 1088-
1095.
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. (2004) Targeted viral delivery of Cre recombinase
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Genomics
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18
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