Electroporation to deliver plasmid DNA into rat dental tissues

Abstract

Background: Delivery of DNA into the target tissues is an important technique in gene function studies and gene therapy. Surgical treatment of tooth eruption disorders, such as impacted third molars, is a major healthcare cost. Because the dental follicle (DF) is essential for regulating tooth eruption, establishment of local gene transfer protocols is needed to determine the effect of various genes on eruption and to develop gene therapy approaches for inducing the eruption of impacted molars.

Methods: Plasmids containing lacZ reporter gene were injected into rat mandibles and then electroporated at the designated settings. Mandibles were collected 24 h after electroporation for X-gal staining to evaluate the transfection efficiency. Tissues were collected at various days post-electroporation to determine the expression of the transgene.

Results: For the DF, depth of injection and pulse number appear to be important. Six pulses can achieve above 80% transfection of the DF at 50 V or 120 V. For alveolar bone (AB) transfection, voltages are important, with 120 V being optimal. Regarding pulse durations, we determined that durations of 20 and 30 ms achieve the maximum transfection in AB and DF, respectively.

Conclusions: The present study demonstrates for the first time the feasibility of electroporation to locally deliver plasmids into dental tissues. Parameters affecting electroporation to deliver plasmids into the dental tissues were optimized. This protocol could be used to deliver short hairpin RNA or genes of interest into the dental tissues to regulate tooth eruption. Thus, it may be possible to develop nonsurgical treatments for inducing the eruption of impacted teeth.

Fig. 1

Plasmids were injected into the mandibles of rat postnatal pups with a syringe attached by a stopper to control the injection depth (A). Instruments used for electroporation in this study included a self-generated forceps electrodes connected to a BTX ECM850 Electro Square Porator. The diagram shows the electrodes clamped on the labial and lingual sides of the tooth crypt for electroporation (B).

Fig. 2

Transfection of rat dental follicle and its adjacent tissues by electroporation with lacZ plasmid. The blue staining indicates that the lacZ gene was delivered and expressed in the dental tissues. Panels A–D are low power micrographs showing different patterns of transfection as seen in the basal part of the molar (A, B); in the coronal part of the molar (C); and around the entire molar (D). Panels E–G are higher power micrographs showing the detailed transfection as seen in the dental follicle (E); in the alveolar bone (F); and in the dental follicle and other dental tissues such as stellate reticulum (G). No staining is seen either in the mandible injected with plasmid, but without electroporation (H), or in the negative control without plasmid injection and without electroporation (I).

Fig. 3

Electroporation to transfect lacZ plasmid into rat dental tissues. (A) Effect of electrode polarity on % transfection of alveolar bone (AB) and dental follicle (DF). Total sample size=23; (B) Rank-sum test to compare the effect of plasmid injection depth on transfection of AB and DF. Note that a significant difference was seen between injection depths of 1.0 and 1.8 mm for transfection of DF as indicated by **. Total sample size= 51; (C) Rank-sum test to compare the effect of pulse durations on transfection of AB and DF. Note that 20 ms duration resulted in the maximum transfection of AB which is significantly higher than durations 30 and 40 ms as indicated by different letters. No significant difference duration effect was detected for transfection of DF. Total sample size=24.

Fig. 4

Effect of plasmid diffusion on transfection of the dental tissues. The plasmids were allowed to diffuse at the times indicated after injection prior to electroporation. Note that the blue staining (arrows) was seen around the molar when the mandible was electroporated immediately (0 min) or 5 min after injection (A, B), but less staining (arrow) was seen when electroporation was conducted 10 or 30 min after injection (C, D).

Fig. 5

Expression of LacZ transgene in alveolar bone and the first molar of rat pups after electroporation to transfect plasmids, as determined by RT-PCR and electrophoresis in agarose gel. Tissue samples were collected at the days indicated post-electroporation. Amplification was seen for both bone and molar samples at all days except day 6 in bone. No amplification was seen in the negative control without cDNA in the PCR. The positive controls using LacZ plasmid and dental follicle cell cDNA indicate the correct amplification of the LacZ and β-actin genes in the PCR reactions.