Origen y significación probables de la mutilación dentaria

Cells: NIH 3T3 seeded in a 6-well plate.

General settings: 6 µg DNA/well. trMAG/DNA w/w ratio: 1.

Biotinylated PEI (bPEI): bPEI/DNA N/P ratio = 8.

Chemically inactivated and biotinylated adenovirus (inact. bAdv): 8.625 x 109 inact. adv particles/6 µg DNA.

Each preparation in HBS.

DNA stock: 48 µg/ml in HBS.

trMAG stock: 1 mg/ml in HBS.

trMAG suspension: 57.6 µg/ml in HBS.

bPEI (25 kDa) stock: 50 µg/ml in HBS.

Inact. bAdv stock: 6.9 x 1010 virus particles per ml in HBS. trMAGs: streptavidinylated trMAG-PEI (trMAG-PEI-Sta).

Controls: same complexes but without magnet and complexes lacking trMAGs and no magnet.

Incubation time: cells were incubated with vectors (with or without magnet) for 15 min.

Vector preparation: bPEI / DNA / inact. bAdv / trMAG-PEI-Sta complexes: 300 µl DNA stock were mixed with 300 µl bPEI stock. Then, 300 µl inact. bAdv stock were added and mixed very gently. Finally, 300 µl trMAG suspension were pipetted to the complexes.

bPEI /DNA / inact. bAdv complexes: 150 µl DNA stock were mixed with 150 µl bPEI stock. Then, 150 µl inact. bAdv stock were added and mixed very gently. Finally, 150 µl HBS were pipetted to the complexes.

X-gal staining: After 24 h, the cells were washed with PBS and subjected to X-gal staining for 45 min.

2.5.7 Magnetofection of other cells

2.5.7.1 HaCaT cells

Cells: HaCaT cells (cell line derived from human keratinocytes, kindly provided by Dr. Martin Mempel, Dermatology, TU Munich, Germany) seeded in 96-well plates. Medium: DMEM without supplements.

General settings: 1 µg DNA/well at PEI containing vectors and 0.1 µg DNA/well at GenePORTER containing vectors.

50 µl transfection volume/well at PEI containing vectors and 100 µl transfection volume/well at GenePORTER containing vectors.

trMAG/DNA w/w ratio: 4. PEI/DNA N/P ratio = 8.

5 µl GenePORTER (Gene Therapy Systems, La Jolla, CA, USA)/1 µg DNA. Each preparation examined in triplicates.

DNA stocks: 8.4 µg/0.140 ml in 0.9 % NaCl for PEI containing vectors and 0.84 µg/0.280 ml in serum-free DMEM for GenePORTER containing vectors.

trMAG stocks: 16 mg/ml in 0.9 % NaCl for PEI containing vectors and 1 mg/ml in serum- free DMEM for GenePORTER containing vectors.

trMAG suspensions: 15.6 µg/0.065 ml in 0.9 % NaCl for PEI containing vectors and 1.56 µg/0.130 ml in serum-free DMEM for GenePORTER containing vectors.

PEI (25 kDa) stock: 8.1 µg/0.130 ml in 0.9 % NaCl.

GenePORTER stock: 3.9 µl/0.260 ml in serum-free DMEM.

trMAGs: trMAG-16/1.

Incubation times: cells were incubated with vectors and magnet for 4h.

Reporter gene assay: luciferase.

Vector preparation: trMAG-16/1 / DNA / PEI complexes: 60 µl DNA stock were mixed with 60 µl trMAG suspension. Finally, 60 µl PEI stock were added to the mixture.

PEI-DNA complexes: 60 µl DNA stock were mixed with 60 µl PEI stock. Finally, 60 µl 0.9 % NaCl were added.

trMAG-16/1 / DNA / GenePORTER complexes: 120 µl DNA stock were mixed with 120 µl trMAG suspension. Finally, 120 µl GenePORTER stock were added to the mixture.

GenePORTER-DNA complexes: 120 µl DNA stock were mixed with 120 µl GenePORTER stock. Finally, 120 µl serum-free DMEM were added to the mixture.

Application of vectors: Fifty µl each of the PEI containing complexes were added to cells kept in 150 µl fresh medium.

Hundred µl each of the GenePORTER containing complexes were added to cells from which the medium was removed before.

2.5.7.2 Primary human keratinocytes

Cells: primary human foreskin keratinocytes (kindly provided by Dr. Martin Mempel, Dermatology, TU Munich, Germany) seeded in 96-well plates. Medium: DMEM without supplements.

General settings: 1 µg DNA/well. trMAG/DNA w/w ratio: 2.

PEI/DNA N/P ratio = 8. Preparation in 0.9 % NaCl.

The various incubation times were examined in triplicates each.

DNA stock: 25.7 µg/0.429 ml in 0.9 % NaCl.

trMAG stock: 5 mg/ml in water.

trMAG suspension: 51.5 µg/0.429 ml in 0.9 % NaCl.

PEI (25 kDa) stock: 26.8 µg/0.429 ml in 0.9 % NaCl. trMAGs: trMAG-16/1.

Controls: 10’ with vectors but no magnet and 4 h with vectors but no magnet (in triplicates each).

Incubation times: 10’ with vectors and 4 h with magnet, 4 h with vectors and 4 h with magnet, 10’ with vectors and 10’ with magnet, 4 h with vectors and only the first 10’ with

magnet. Incubation with vectors and application of a magnetic field always started simultaneously.

Reporter gene assay: luciferase.

Vector preparation: trMAG-16/1 / DNA / PEI complexes: 390 µl DNA stock were mixed with 390 µl trMAG suspension. Finally, 390 µl PEI stock were added to the mixture.

2.5.7.3 RIF-1 cells

Cells: RIF-1 cells (mouse radiation-induced fibrosarcoma cell line, kindly provided by Ellen Kolbe, Experimental Oncology, TU Munich, Germany) seeded in a 96-well plate. Medium: DMEM with supplements.

General settings: 1 µg DNA/well. trMAG/DNA w/w ratio: 4.

PEI/DNA N/P ratio = 8. Preparations in 0.9 % NaCl.

Each preparation was examined in triplicates.

DNA stock: 12.6 µg/0.210 ml in 0.9 % NaCl.

trMAG stock: 16 mg/ml in water.

trMAG suspension: 33.6 µg/0.140 ml in 0.9 % NaCl.

PEI (25 kDa) stock: 8.8 µg/0.140 ml in 0.9 % NaCl. trMAGs: trMAG-16/1.

Control: Incubation with the standard vector PEI-DNA for 2 h (in triplicates).

Incubation times: cells were incubated with magnetofectins for 30 min.

Reporter gene assay: luciferase.

Vector preparation: trMAG-16/1 / DNA / PEI complexes: 65 µl DNA stock were mixed with 65 µl trMAG suspension. Finally, 65 µl PEI stock were added to the mixture.

trMAG-16/1 / DNA complexes: 65 µl DNA stock were mixed with 65 µl trMAG suspension. Finally, 65 µl 0.9 % NaCl were added to the mixture.

PEI-DNA complexes: 65 µl DNA stock were mixed with 65 µl PEI stock. Finally, 65 µl 0.9 % NaCl were added to the complexes.