Heparin

Mitotic chromosomes were prepared from nonsynchronized cultures of peripheral blood lymphocytes collected on Heparinized tubes. Whole blood (1 ml) was cultured for 72 h in a medium consisting of 9 ml RPMI (Gibco), 20% fetal bovine serum, and 500 IU Heparin (Sanofi), and stimulated with 0.2 ml pokeweed mitogen (Gibco). Hypotonic treatment (10 ml 1/6 calf serum) was followed by prefixation and fixation in ethanol: acetic acid (3:1). Chromosome preparations were spread on cold wet slides and air‐dried. Slides were stained with 3% Giemsa solution. For each individual, the number of chromosomes of at least ten cells was counted.

The complexation of mRNA within formulations was assessed using a gel retardation assay for electrophoresis. Agarose gel (1%) was prepared in Tris–borate–EDTA (TBE) (1×) buffer-containing ethidium bromide staining (EtBr, Genesee Scientific, San Diego, CA, USA). Samples were mixed with 2× loading dye (2× solution of 95% formamide, 18 mM EDTA, and 0.025% SDS, xylene cyanol, and bromophenol blue; Invitrogen, Carlsbad, CA, USA) and a volume corresponding to 100 ng (or equivalent volume for controls) of mRNA was loaded in each well. The electrophoresis process was run for 17 min at 100 V and the gel was observed in UV-Visible.
To dissociate mRNA from vectors and check its denaturation, a treatment was performed on samples prior to deposition. Briefly, formulation samples were treated—firstly with heparin (Sanofi-Aventis, Ploermel, France) for 1 h at RT, and secondly with proteinase K (NEB, Evry, France) for 30 min at 56 °C. Samples were then loaded into the gel and analyzed as described above.

Aortas were collected as follows. After euthanasia, 2500 UI heparin (Sanofi, Paris, France) were injected in the heart to prevent blood coagulation. The heart and aorta were then flushed with 10 mL phosphate buffered saline using a syringe driver at 2 mL/min for control mice and 1.5 mL/min for diabetic mice (Harvard Apparatus, Holliston, MA, USA) to remove residual blood. The aorta was further prefixed by injecting 5 mL of 4% formalin with the same flow. Then, 5 min after the end of the fixative solution injection, 6 mL of 1% low melting agarose (Fisher Bioreagents, Waltham, MA, USA, BP165-25) were injected (2 mL/min control, 1.5 mL/min diabetic mice) to keep the aorta opened and to prevent collapse during dehydration and inclusion steps. The system was then left to rest at room temperature for 5 min so that the agarose solidified as a gel in the lumen and exerted a counterpressure to avoid collapse. The heart and aorta were collected with surrounding tissues and placed in a cassette where the aorta was held at both ends by a wire to keep it straight. Finally, the samples were fixed in 4% formalin for 24–48 h, dehydrated, and embedded in paraffin. The final samples were about 4-cm-long paraffin rods containing the heart and aorta.

Mice were deeply anesthetized with sodium pentobarbital (i.p., 100 mg/kg, Sanofi) and perfused transcardially with a solution containing 0.9% NaCl and heparin (Sanofi-Synthelabo), followed by 4% paraformaldehyde in phosphate buffer, pH 7.3. Brains were removed and postfixed by incubation in the same fixative at 4 °C overnight. Tissues were cryoprotected by incubation in 30% sucrose in PBS for 24 h. Immunostaining was performed on 40- or 60-µm-thick coronal brain sections obtained with a vibrating microtome (VT1000S, Leica). Nonspecific staining was blocked by 0.2% Triton, 4% bovine serum albumin (Sigma-Aldrich), and 2% goat serum and free-floating slices were then incubated with the following primary antibodies at 4 °C overnight: rabbit anti-DCX (Abcam, ab 18723), rabbit anti-Ki67 (Abcam, ab16667), and mouse anti-NeuN (Millipore, MAB377). Secondary antibodies (Alexa Fluor-conjugated secondary antibodies, Molecular Probes) were then incubated at room temperature. 4,6-Diamidino-2-phenylindole (1 µg/ml) was used as a nuclear stain. EdU was visualized using the Click-iT reaction coupled to an Alexa Fluor® azide following the instructions of the manufacturer (Molecular Probes).

Electroporated animals aged between postnatal day P78 and P129 were anesthetized with ketamin 100 mg/kg and xylazin 10 mg/kg and intracardiacally perfused with first 0.1 mL of heparin (5000 U.I/mL, Sanofi, Paris, France), then an aqueous solution of 4% w/v paraformaldehyde (PFA) (CliniSciences) and 0.5% glutaraldehyde (GA) (Clinisciences, Nanterre, France) in 0.1 M phosphate-buffered saline (PBS). The fixative solution was made extemporaneously and kept at ice-cold temperature throughout the perfusion. The perfusion was gravity-driven at a flow rate of about 0.2 ml/s, and the total perfused volume was about 100 ml per animal. Brains were collected and postfixed overnight at 4°C in a 4% PFA solution. Coronal brain sections with 30-μm thickness were obtained using a vibrating microtome (Leica VT1200S).

The complexation of mRNA onto LP and LP_auto was evaluated using an electrophoretic mobility shift assay. To this aim, a 1% agarose gel was prepared in Tris–borate–EDTA (TBE) 1× buffer, and ethidium bromide (EtBr) was added as staining. Formulations were either treated or not with heparin (Sanofi-Aventis, Ploermel, France) at RT for 30 min plus with proteinase K (NEB, Évry-Courcouronnes, France) at 56 °C for 15 min. The gel was then loaded with samples mixed with 2× loading dye (2× solution of 95% formamide, 18 mM EDTA, 0.025% SDS, xylene cyanol, and bromophenol blue; Invitrogen, Carlsbad, CA, USA) containing 100 ng of mRNA per well. The electrophoresis was performed for 17 min at 100 V, and the stained mRNA bands were visualized on an ultraviolet transilluminator and digitalized.