crc press - E-Lib FK UWKS

Penetratins 45
FCS (10%) or horse serum (10%). Internalization can be done on cells grown on
plastic or glass (coated or not with polylysine, laminin, collagen).
The only crucial point is that the coupled product must be diluted in the culture
medium before its addition on cells. Because of the rapidity of the internalization
process, direct addition of the concentrated product on cells can lead to a heterogeneous
distribution among cells.
2.5.2.2.1 Oligonucleotide Internalization
The following protocol is appropriate for antisense oligonucleotide experiments, but
optimal concentration and incubation time must be determined in each case. Internalization
and intracellular distribution of the cargo can be visualized by using a
labeled oligonucleotide. Doubly modified oligonucleotides containing an SH modification
at the 3′ end and a fluorescein or a biotin at the opposite end are commercially
available.
Steps
1. Grow the cells in appropriate medium. The number of cells is an important
parameter; 10 5 cells/well in a 24-well plate at the time of coupled oligonucleotide
addition is usually appropriate.
2. Dilute the coupled oligonucleotide in 500 µL fresh culture medium (1 µ M
to 1 nM final) and add it to the cells after removal of the culture medium
(if present, DMSO concentration should not exceed 0.1%).
3. Incubate the cells in normal culture conditions until needed. The effect
of the oligonucleotide can be evaluated a few hours after its addition (see
Section 2.4). Because of the limited stability of intracellular oligonucleotides,
biological effects should be evaluated within 2 days.
4. If culture times longer than 1 day are required after treatment, repeat the
treatment every 24 h. Proceed as in step 2.
2.5.2.2.2 Peptide Internalization
The ability to internalize small peptides is a promising feature of homeodomainderived
vectors. Internalized peptides can exert direct effects on biological functions,
which can be monitored as early as a few minutes after their addition; 31 the effects
can last up to 8 days. 43 In contrast with oligonucleotides, peptide internalization can
be greatly affected by its amino acid sequence. Even if the vector has been used
successfully with different kinds of peptides, it is preferable to test internalization
in each case. In order to be visualized, cargo peptide can be labeled as described
for penetratin (Section 2.5.1.1).
Solutions
1. 1 M MgCl 2: dissolve 20.3 g MgCl 2·6H 2O in 80 ml distilled water. Adjust
to 100 ml with distilled water.
2. 10× deoxyribonuclease I (300 µg/ml): dissolve 1 mg DNase I in 3.2 ml
PBS. Add 67 µl of 1 M MgCl 2. Filtrate, sterilize, and store at –20°C in
aliquots.