In Vivo Gene Transfer to Mouse Spermatogenic Cells: MATERIALS AND METHODS(3)

MATERIALS AND METHODS(3)In Vivo EP

Mice were anesthetized, and the testis was exposed under a dissecting microscope. A small (2-3-mm) incision was made in the tunica, and then 20 ^l per testis of plasmid DNA solution, to which 0.04% Trypan blue dye had been added to monitor the accuracy of the injection, was injected into the seminiferous tubules (intratubular injection) or interstitial space of the testis (intratesticular injection) using injection glass pipettes (tip 30- to 40-^m in diameter). For the latter case, injections were made at three sites in each testis. After DNA injection, EP was performed with an electroporator (Electrosequare Porator T820; BTX, San Diego, CA). Testes were held between a tweezers-type electrode, and square electric pulses were applied eight times at 20-50 V with a constant time of 50 msec according to the procedure of Muramatsu et al.. These treatments produced no noticeable damage on testes at histopatholog-ical observation. After EP treatment, the skin was stitched, and the mice were raised until analysis. buy cheap antibiotics

Histochemical Analysis

Histochemical staining of p-galactosidase derived from transferred plasmid was performed as described previously. The testes were fixed for 1-2 h in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3), rinsed three times in phosphate buffer containing 2 mM MgCl2 and 0.02% NP-40, and stained for 1-2 h at 37°C in the same buffer containing 0.1% 5-bromo-4-chloro-3-indolyl-p-D-galacto-pyranoside (X-gal; Sigma), 5 mM K3Fe(CN)6, and 5 mM K4Fe(CN)6. For light microscopic observation, serial paraffin sections (8 or 15 ^m) of the testes were prepared and counterstained with 0.5% eosin Y.

Dual Luciferase Assay

The dual luciferase reporter assay was performed according to the manufacturer’s instructions (Promega). At 18 h after transfection, the whole testis was homogenized in 0.8 ml of ice-cold lysis buffer, and the crude lysates were clarified by centrifugation (12 000 rpm, 10 min) at 4°C. Ten microliters of the supernatant was first mixed with 100 of luciferase substrate to assay firefly luciferase reporter activity for 20 sec using a luminometer (Lumat LB 9501, Berthold, Wildbad, Germany), and then Renilla luciferase control activity was measured for 20 sec after addition of 100 ^l of Stop & Glo buffer (Promega) to the reaction.

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