scientificprotocols authored over 6 years ago
Authors: Joanna Szczurkowska, Andrzej W. Cwetsch, Marco dal Maschio, Diego Ghezzi,
Gian Michele Ratto & Laura Cancedda
One of the challenges for modern neuroscience is to understand the role of specific genes in the determination of cellular fate, and in the formation and physiology of neuronal-circuits. Techniques for genetic manipulation in vivo such as in utero electroporation are fundamental tools to address these issues. Here, we describe an established protocol for in utero electroporation in mouse and rat for reliable targeting of the hippocampus, the motor, prefrontal, and visual cortices, and the Purkinje cells of the cerebellum. The method is based on an electroporation configuration entailing commonly used forceps-type electrodes plus an additional third electrode. This configuration allows highly consistent direction of the electric field to the different neurogenic areas by simple and reliable adjustment of relative positions, polarities and/or dimensions of the electrodes. More than 70% of electroporated embryos survive to postnatal ages and around 60-90% express the electroporated vector, depending on the targeted area. By a single electroporation episode, the protocol enables for symmetric transfection in both brain hemispheres. The procedure requires 4 hours of preparation on the first day and it lasts 1 hour, including a surgery time of 30 mins, on the second day.
In utero electroporation was first described in 2001 as a simple and quick procedure to efficiently perform in vivo genetic manipulation of pyramidal neurons of the rodent somatosensory cortex (1-3). The technique takes advantage of the fact that, by addressing neural progenitors at the epithelium of the ventricular system, one can genetically manipulate specific populations of newborn neurons that will migrate to different brain areas (5). Therefore, in utero electroporation has theoretically tremendous potentiality for addressing cells in many brain regions by targeting progenitors at their diverse neurogenic areas at the proper developmental stage. Over the past ten years, the number of laboratories using the technique to target the somatosensory cortex has risen exponentially, as confirmed by the increasing number of publications in the field (4). Nevertheless, it is clear that the experimental conditions required to target other brain areas are not reliable, as demonstrated by the incredibly low number of publications with in utero electroporation targeting brain locations other than the somatosensory cortex (4). Moreover, some brain areas theoretically attainable have never been successfully electroporated, likely because of the physical impossibility to target the corresponding neurogenic regions with the electric field generated by two forceps-type electrodes, as initially indicated for the technique. In the present manuscript, we describe an in utero electroporation configuration based on the usage of three electrodes, which allows easy and exceedingly reliable transfection at brain locations only sporadically targeted before, just by varying the relative position, polarities and/or dimensions of the electrodes.
Advantages of the three-electrode configuration
The tripolar configuration for in utero electroporation presents the following advantages:
It allows extremely easy transfection of many different brain areas (hippocampus, visual cortex, motor cortex, prefrontal cortex, cerebellum), with a degree of reliability comparable to that attained in the somatosensory cortex with the bipolar electrode (BOX1).
It allows transfection efficiency higher than the conventional two-electrode configuration, and it is proved to enable for the use of lower voltages for comparable outcomes (4,6).
It allows symmetric electroporation of both brain hemispheres by a single electroporation episode in virtue of the symmetrical electrical field generated by the three electrodes (BOX4)(4).
Limitations of the three-electrode configuration
The main drawback of the three-electrode configuration is that it requires two operators during the electroporation phase of the procedure. One person will hold the embryo and the forceps-type electrodes and another person will hold the third spare electrode. Nevertheless, a trained operator and an ergonomic design of the tool can overcome this limitation (4,6).
Preparation of tools. TIMING 4hrs, 1d before surgery
To electroporate different brain structures use the following electrode configurations:
A. HIPPOCAMPUS
Use the forceps-type electrodes connected by a Y- connector to the positive pole and gently grab both sides of the embryo’s head. Connect the third electrode to the negative pole placed at 0o with respect to the horizontal plane right above bregma (Fig. 7)
B. MOTOR CORTEX
Use the forceps-type electrodes connected by a Y- connector to the negative pole, and gently grab both sides of the embryo’s head. Connect the third electrode to the positive pole, and place as for electroporation of the hippocampus (Fig. 8)
C. PREFRONTAL CORTEX
Use the forceps-types electrode connected by a Y-connector to the negative pole and place it on both sides of the embryo’s head. Connect the third electrode to the positive pole, and place it on the front of the embryo’s head (Fig. 9)
D. VISUAL CORTEX
Use the tweezer type electrode connected by a T- connector to the negative pole and place it on both sides of the embryo’s head. Connect the third electrode to the positive pole, and place it right on (rat) or below (mouse) lambda (Fig. 10)
E. CEREBELLUM
Use the forceps-type electrodes connected by a T- connector to the negative pole and place it on both sides of the neck at the level of the fourth ventricle. Connect the third electrode to the positive pole, and place it on top of the fourth ventricle (Fig. 11)
The procedure requires 4 hours of preparation on the first day and it lasts 1 hour on the second day.
Detailed timing of single procedures:
Neuronal fate of transfected progenitors and migration of newborn-neuron to different brain areas can be easily followed when in utero electroporation of a fluorescent protein is performed (4,6). Bilateral transfection of both brain hemispheres is achievable with a single electroporation episode, allowing for efficient electrophysiological and behavioral experiments (BOX 4). Comparison of possibly different functions of a same gene in various brain areas characterized by different cellular contexts is possible thanks to the high reliability of transfection at various brain locations (BOX 1). Transfection efficiency for different brain areas depends on how accurately one follows this protocol. Accurate matching of the dimension of the forceps-type electrodes to that indicated in the protocol is crucial for high reliability of electroporation (BOX 1). Increase of the survival rate can be achieved by lowering the electroporation voltage during surgery, due to the higher efficiency of the three-electrode configuration in comparison to bipolar in utero electroporation4. Survival rate is mostly improved by pup fostering and after-surgery care, as indicated in the protocol and boxes. Moreover, the high efficiency and reliability of transfection allows for reduced number of dams required (in agreement with EU guidelines), and decreased costs of animal housing. We conclude that the three-electrode electroporation offers a conceptual advance to the field by paving the way to targeting brain areas never electroporated before by simply varying the number, together with the polarities and dimensions of the electrodes.
We thank Francesca Managò (IIT) for her idea to use a cell scraper as the holder of the third custom-made electrode and Giacomo Pruzzo (IIT) for technical help in crafting it.
Figure 1: Equipment for in utero electroporation.
Figure 2: Three-electrode configuration for in utero electroporation.
a) Tripolar in utero electroporation configuration entails two conventional forceps-type electrodes connected to a single polarity by a Y-connector and an additional third custom-made electrode. Scale bar: 5cm. (b) High magnification of Y-connector for connection of commercial forcepsr-like electrodes to a same pole. Scale bar: 1cm. (c) Side (left) and front (right) views of additional third electrodes for electroporation of rat and mouse. Third electrodes are made from a commercial cell scraper and a platinum plate. Optimal sizes of platinum plates are reported in the figure. Scale bars: 5mm.
Figure 3: Surgical tools for in utero electroporation.
(a) Ring forceps (1); Shark-tooth tissue forceps (2); Scissors with flat shanks –angular (3); Scissors with flat shanks – straight (4); Olsen-Hegar needle holders with scissors (5); Scalpel (6); Scalpel blade (7). (b) Surgical tools, plastic connectors and gauzes in self-sealing autocavable pouch.
Figure 4: DNA injection in the embryo by a commercial needle.
Examples of a mouse embryo (E15.5) during (a) and after (b) DNA injection in the lateral ventricle by the syringe needle filled with DNA and Fast Green dye for vizualization (blue). Note the green half-moon shaped spot, indicating complete filling of the lateral ventricle (arrow). (c) DNA injection of a rat embryo (E14.5) in the fourth ventricle. White arrow indicates filling of the fourth ventricle. (d) Syringe needle connected to the tubing of the pico-pump by a plastic connector. Scale bar: 1cm. (e) High magnification of commercial plastic connectors that connects the syringe needle and the picospritzer. Scale bars: 1cm and 5mm.
Figure 5: The exposed uterus is wet with warm and sterile PBS before electroporation.
Figure 6: Example of in utero electroporation of mouse embryos (E15.5) with the usage of the additional third electrode and different sizes of forceps-type electrodes.
Addition of the third electrode and usage of different sizes of the forceps-type electrodes increase in utero electroporation efficiency. Scale bar: 5 mm.
Figure 7: Top (left) and front (left) views of the three-electrode configuration for in utero electroporation of mouse and rat embryos in the hippocampus.
– and + indicate polarities of the additional third and forceps-type electrodes, respectively. White dotted lines indicate coronal and sagital sutures on the scull. The white dot indicates Bregma and the black dot indicates Lambda. Scale bars: 3mm (mouse), 5mm (rat).
Figure 8: Top (left) and front (left) views of the three-electrode configuration for in utero electroporation of mouse and rat embryos in the motor cortex.
Figure 9: Front views of three-electrode configuration for in utero electroporation of mouse (left) and rat (right) embryos in the prefrontal cortex.
Figure 10: Top views of the three-electrode configuration for in utero electroporation of mouse (left) and rat (right) embryos in the visual cortex.
Figure 11: Side (left) and back (right) views of the hree-electrode configuration for in utero electroporation of rat embryos in the cerebellum.
Table 1: Proper isoflurane and oxygen levels for in utero electroporation on mouse and rat.
! CAUTION All experiments must be performed in accordance with relevant institutional and governmental guidelines and regulations.
Table 2: Standard parameters for tripolar in utero electroporation of mouse and rat.
Table 3: Troubleshooting
Boxes: Boxes
High-performance and site-directed in utero electroporation by a triple-electrode probe. Marco dal Maschio, Diego Ghezzi, Guillaume Bony, Alessandro Alabastri, Gabriele Deidda, Marco Brondi, Sebastian Sulis Sato, Remo Proietti Zaccaria, Enzo Di Fabrizio, Gian Michele Ratto, and Laura Cancedda. Nature Communications 3() 17/07/2012 doi:10.1038/ncomms1961 doi:10.1109/EMBC.2013.6609825
Joanna Szczurkowska, Andrzej W. Cwetsch, Marco dal Maschio, Diego Ghezzi & Laura Cancedda, Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia, via Morego, 30, 16163 Genova
Gian Michele Ratto, Center for Nanotechnology Innovation (NEST), Institute Nanoscience, National Research Council, Piazza San Silvestro 12, Pisa, Italy & Institute of Neuroscience, National Research Council, Piazza San Silvestro 12, Pisa 56127
Correspondence to: Laura Cancedda ([email protected])
Source: Protocol Exchange (2013) doi:10.1038/protex.2013.089. Originally published online 2 December 2013.