1Center for Neurologic Diseases, Brigham and Woman's Hospital and Harvard Medical School, 2Department of Physiology and Neurobiology, University of Connecticut
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Rice, H., Suth, S., Cavanaugh, W., Bai, J., Young-Pearse, T. L. In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons. J. Vis. Exp. (44), e2103, doi:10.3791/2103 (2010).
In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.
The basic protocol for in utero electroporation has been described in detail in another JoVE article from the Kriegstein lab 1, 2. This technique was originally described in the Osumi lab 3 and our protocol is based upon one developed in the LoTurco lab 4. We will provide an overview of the our protocol for in utero electroporation of rat embryos, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.
1. In utero Electroporation
2. Culturing Electroporated Cortical Neurons
3. Analyzing Neuronal Process Outgrowth
4. Representative Results
We have found that Sprague Dawley litter size ranges between 6 and 14 embryos. We usually electroporate all of the embryos. Each embryo can be electroporated with a different combination of DNAs. However, we usually electroporate at least four brains with the same condition and pool these brains before dissociating and plating.
We have found that with this technique approximately 75% of electroporated brains are targeted to the desired region of the cortex, whether that be dorsal medial or ventral lateral cortex (Figure 1). In addition, we have found early electroporations at E13-14 target deep layer neurons such as Tbr1 positive layer VI neurons, while later electroporations target CTIP2 positive, TBR1 negative layer V cells, and still later electroporations target Brn2 positive layer II/III cells. An excellent description of different markers and explanation of neuronal subtype specification in the cortex in found an article by Moleneaux et al 7. Figure 2 shows coronal sections of brains electroporated at either embryonic day 15.5 or 17.5 and harvested at postnatal day 5. Shown in red is immunostaining for Oct6. You can immunostain for markers in culture to confirm what cell layer populations you have targeted. We have found that you can expect to target the same cell layer population of cells in every embryo of the same litter (in other words, the targeting depends upon the embryonic timepoint rather then on other technical variations).
In culture, the percent of cells that are GFP positive can range widely depending on how conservative you are when dissecting out the GFP positive region (Figure 3). However, even when we are very conservative and dissect out only the GFP positive patch of cells, the highest percentage that we observe is 5-10% - although you are dissecting the region of the cortex that was electroporated, cells in only one layer will be targeted. This low transfection effciency is helpful in identifying which processes belong to the electoporated cell that you are analyzing. Plating cells at this higher density contributes to having heathier cultures, however, it is difficult to discern which process belongs to which cell body in the GFP negative cells (Figure 3).
If you have trouble seeing all of the fine processes of the electroporated cells, you can either increase the concentration of GFP DNA that you are electroporating to increase expression of GFP, or you can immunostain the dissociated cells using an anti-GFP antibody (from Invitrogen) along with a Cy2 secondary antibody.
Figure 1. E15.5 Sprague-Dawley rats were electroporated with GFP plasmid and harvested three days later. Based upon the placement of the electrodes, different regions of the cortex will be targeted. A-F show GFP fluorescence in whole brains.
Figure 2. E15.5 (A) or E17.5 (B) Sprague-Dawley rats were electroporated with GFP plasmid and harvest at postnatal day 5. Brains were fixed, sectioned coronally using a vibratome (100 micron sections), and immunostained for Oct6 (red). A and B show confocal images of immunostained sections.
Figure 3. E15.5 Sprague-Dawley rats were electroporated with GFP plasmid. 24 hours following electroporation, brains were harvested and GFP-positive, electroporated regions were dissected and dissociated, as described in the video. After 3 days in vitro, cells were fixed and immunostained for bIII-tubulin (red) and staining nuclei with DAPI (blue) (A,C). The length of the longest neurite was measured using Axiovision software (B,D).
In vitro study of primary neuronal cultures allow for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or misexpression constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex.
Targetting of specific layers:
When you inject DNA into the lateral ventricle and electroporate, only the cells immediately lining the lateral venrtricle are targeted: these cells are the radial glial progenitor cells of the neocortex, as well as cells that have just undergone their terminal mitosis. Interestingly, when examined in the days following electroporation, the cells targeted match the pattern of birthdated cells. In other words, cells that are born , that is to say those that undergo their terminal mitosis on the day of electropoartion, are the cells that are targeted. Since radial glial cells also are present at the ventricular surface, one might hypothesize that these cells would be targeted, and that all of the progeny from these cells would also be targeted. However, this is not the case, later generations of cells do not express GFP. It may be that multiple rounds of division in the radial glial cells dilutes out the plasmid.
This method is an excellent way to examine the effects of gene knock down via electroporation of shRNA contructs, as well as by misexpression of cDNA constructs. We are applying this techniqe to the study of genes involved in neurodegeneration and psychiatric disease. Through this technique, we introduce both wild type and mutant forms of genes critical in these diseases, and examine the effects on neuronal morphology. In addition, we can examine the effects of mutation or alternative splice variant expression in the absence of the endogenous gene product by co-electroporating the cDNA and the shRNA contruct. Co-electorporation also can be utilized to look at genetic interactions between two gene products: by knocking down multiple genes and by attempting to rescue effects of knock down of one gene product with other gene products 8, 9.
No conflicts of interest declared.
The authors would like to thank Joseph LoTurco and Dennis Selkoe for helpful discussions on this technique. The authors thank the donors of the American Health Assistance Foundation, for support of this research.
|10X Hanks’ Balanced Salt Solution (HBSS) (Ca+2 /Mg +2 free)||GIBCO, by Life Technologies||14185-052|
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|Dulbecco’s Modified Eagle Medium (D-MEM)||GIBCO, by Life Technologies||11960-051|
|Fetal Bovine Serum||Sigma-Aldrich||F4135|
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