These issues can be circumvented by using a bulk electroporation approach. labeling in any brain area where bulk electroporation is possible. Unlike juxtacellular single-cell electroporation methods, CREMSCLE relies exclusively on the bulk electroporation technique, circumventing the need to precisely position a micropipette next to the target cell. Compared with viral transduction methods, it is fast and safe, generating high levels of expression within 24 h of introducing non-infectious plasmid DNA. In addition to increased efficiency of single-cell labeling, we confirm that CREMSCLE also allows for efficient co-expression of multiple gene products in the same cell. Furthermore, we demonstrate that this method is particularly well-suited for labeling immature neurons to follow their maturation over time. This approach therefore lends itself well to time-lapse morphological studies, particularly in the context of early neuronal BCIP development and under conditions that prevent more difficult visualized juxtacellular electroporation. situations where the targeted cells are hard to visualize under a microscope or so sparsely distributed that blind electroporation attempts are unlikely to succeed. Additionally, the success rate of SCE is usually greatly dependent on micropipette tip shape. Optimization of tip shape requires a process of trial-and-error, which for DNA plasmid delivery cannot provide immediate reliable opinions until the next day when protein expression is (or is not) evident. An alternative to SCE is usually bulk electroporation, which takes advantage of the same principles as SCE for delivery of genetic material into cells, but instead of delivering plasmid and current through the same pipette, it utilizes large plate electrodes that are positioned on opposite sides of the structure targeted for transfection and simple pressure injection to deliver plasmid into the extracellular space between the electrodes (Muramatsu et al., 1998; Falk et al., 2007). This method permits the efficient transfection of multiple plasmids or other charged materials just like SCE, but instead of targeting only one cell it is used to target many BCIP cells within larger tissue volumes. One common example of this technique is usually electroporation, in which plasmid is usually injected into the brain ventricles of embryonic animals and electroporation pulses are delivered through forceps-like paddle electrodes that bracket the uterus to generate an electric field within the brain of the embryo (Tabata and Nakajima, 2001; Shimogori and Ogawa, 2008). The obvious advantage of this approach is usually that it does not require obvious visualization or precise positioning of the electrode and is therefore applicable in nearly any tissue. In the current paper, we describe CRE-Mediated Single-Cell Labeling by Electroporation (CREMSCLE), an innovative method that utilizes bulk electroporation to achieve the benefits of single-cell labeling for time-lapse imaging. CREMSCLE entails a binary co-expression approach that takes advantage of the ability of extremely low levels of Cre recombinase protein to edit many copies of a plasmid made up of a neomycin quit cassette flanked by loxP sites that has been inserted into the 5 end of the open reading frame of a gene of interest. This cre-mediated editing event effectively releases translation suppression of the downstream gene of interest. By using this binary approach, we show that co-electroporation of high concentrations of plasmid made up of a gene of interest preceded by the quit cassette, together with extremely low amounts of plasmid encoding Cre recombinase, results in high levels of gene expression in very sparsely distributed individual cells, which constitutes ideal cell labeling conditions for live imaging. We previously published an application of this method to express EGFP in individual retinal ganglion cells in neonatal mouse eyes (Dhande et al., 2011). Here, using the tadpole, which permits easy access for electroporation and visualization of fluorescent protein expression, we compare and contrast CREMSCLE with SCE Rabbit polyclonal to EGFR.EGFR is a receptor tyrosine kinase.Receptor for epidermal growth factor (EGF) and related growth factors including TGF-alpha, amphiregulin, betacellulin, heparin-binding EGF-like growth factor, GP30 and vaccinia virus growth factor. and demonstrate its effectiveness for sparse co-expression of multiple gene products in the same cells. Materials and Methods Animal Breeding and Husbandry All animal experiments were approved by the Montreal Neurological Institute (MNI) Animal Care Committee in accordance with the guidelines of the Canadian Council on Animal Care. Tadpoles were bred by HCG-induced mating of albino frogs (NASCO) in the MNI Animal Care Facility. Embryos were then reared with regular answer changes in bowls made up of Modified Barth Answer with HEPES (MBS-H) buffer. Constructs pCAG-Cre, pCALNL-EGFP, pCALNL-DsRed are a nice gift from BCIP T. Matsuda & C. L. Cepko and are currently available through Addgene (plasmids 13775, 13770, 13769). pEGFP-N1 was from Clontech. mCherry was a nice gift from Dr. Roger Tsien. All plasmids were produced in DH5a qualified cells (Life Technologies) and purified using BCIP endotoxin-free maxiprep packages (Qiagen). Bulk Electroporation Albino tadpoles at stage 44C46 according to.