Three puncture sites used for in utero electroporation show no significantly different negative impacts during gene transfer into the embryonic mouse brain
Introduction
As many new genes are identified by genome projects, the study of the function and the network activity of these genes in vivo becomes a key problem. There are multiple ways to manipulate genes in vivo. Transgenic and gene targeting techniques have the ability to change specific genes that are then stably transmitted to the next generation [7]. Recombinant viruses and biolistic gene guns have been used to deliver genes to in vivo tissues. However, these methods have certain limits. For example, the process of producing transgenic, gene-targeted mice and recombinant viruses is time-consuming and strenuous. Moreover, it is difficult to express the gene of interest precisely at a specified time and location, as the transcriptional regulation of genes is complicated. Thus, in utero electroporation, as a quick and easy method, has been favored by more and more researchers, and the use of this technique will greatly promote the understanding of gene function and networks in the brain.
For electroporation, after introducing the appropriate vectors into the ventricle of embryonic mouse brain, an electric field is applied to the proper extra-uterine position, and the vectors move toward the positive electrode and then to the targeted specific regions. We can study the migration and differentiation of the cells to analyze the functions of genes. Compared with other DNA transfer methods, in utero electroporation method has several advantages: it is a highly efficient, spatially and temporally specific technique and can be used to transfect multiple genes into one cell [14]. Furthermore, in utero electroporation can be used similarly to mitotic birth dating. Following this principle, different electroporation times can lead to expression in specific cell types [8]. The in utero electroporation method has also been used to study the gain of function and loss of function of some genes [4], [17]. The application of this method contributes to illuminate the molecular mechanisms through identification and characterization of the mislocalization of neurons or other cells in brain development and pathophysiology [5], [16]. Therefore, in utero electroporation is playing an increasingly important role in neuroscience. Scientific experiments require following a strict protocol, and small details determine success or failure. For electroporation, essential details include the following: tip diameter of the micropipettes, the puncture site, the voltage and pulse duration of the electric pulse, the clip position of the paddle electrodes, the operation time, DNA extraction, and the concentration and quality of vectors. All these elements will influence the results to a certain extent.
Although the embryonic head can be seen through the uterine wall, it is still difficult to inject DNA precisely into the region of interest, particularly into the diencephalon and ventral parts of the embryonic brain. The selection of puncture sites is a key event. Common puncture sites include the following: injection near the posterior fontanel, at about 1 mm; injection along the antero-posterior axis into the cephalic ventricle; and injection near the midpoint between the anterior fontanel and posterior fontanel, at about 3 mm. Different laboratories have their own methods. Beginners struggle with how to choose the injection position. This article studies the different influences on cortical development when injection is performed at different sites.
Section snippets
Animals
We used CD1 (ICR) mouse, which was purchased from the Vital River Company (Beijing, China). All animal handling protocols were approved by the local Institutional Animal Care Committee.
Plasmid
The plasmid contained a GFP/DsRed reporter gene that is downstream of the CAG promoter (Addgene 11150, Addgene 11151). The plasmids were purified using the EndoFree Plasmid Kit (12362, Qiagen).
In utero electroporation
The in utero electroporation was performed with an electroporator (ECM830, BTX) using five pulses of 45 V with 1 s
Results
To obtain optimal results and decrease the death rate of the fetuses, many details must be paid attention to. Firstly, the preparation of the micropipettes is very important. The diameter, the angle and the smooth degree of the pinpoint may be the key that determines success or failure. For example, if the pinpoint is too thick, it can lead to leakage of cerebrospinal fluid (Fig. 1A-a’); if the pinpoint is too thin, the micropipette is easy to block; if the pinpoint has no angle (Fig. 1A-b’) or
Discussion
To our knowledge, this is the first study to investigate effects of injection sites during in utero electroporation. The major findings of the present study were as follows: (i) there is no statistical significant differences between the three puncture methods on death rate of embryos and GFP-positive staining; (ii) there is no statistical significant differences between the three puncture methods on area and thickness of the cortex; (iii) there is no statistical significant differences between
Acknowledgments
This study was funded by Military Medical Project (BWS11J002, BWS12J010). We thank Elsevier Language Editing (Project nr: 43672) for assisting in the preparation of this manuscript. The authors would like to thank all the anonymous reviewers for their valuable comments on how to improve the quality of this paper.
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These authors contributed equally to this work.