Falk J et al. (2007), Electroporation of cDNA/Morpholinos to targeted...

XB-ART-36524

BMC Dev Biol 2007 Sep 27;7:107. doi: 10.1186/1471-213X-7-107.

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Electroporation of cDNA/Morpholinos to targeted areas of embryonic CNS in Xenopus.

Falk J , Drinjakovic J , Leung KM , Dwivedy A , Regan AG , Piper M , Holt CE .

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BACKGROUND: Blastomere injection of mRNA or antisense oligonucleotides has proven effective in analyzing early gene function in Xenopus. However, functional analysis of genes involved in neuronal differentiation and axon pathfinding by this method is often hampered by earlier function of these genes during development. Therefore, fine spatio-temporal control of over-expression or knock-down approaches is required to specifically address the role of a given gene in these processes. RESULTS: We describe here an electroporation procedure that can be used with high efficiency and low toxicity for targeting DNA and antisense morpholino oligonucleotides (MOs) into spatially restricted regions of the Xenopus CNS at a critical time-window of development (22-50 hour post-fertilization) when axonal tracts are first forming. The approach relies on the design of "electroporation chambers" that enable reproducible positioning of fixed-spaced electrodes coupled with accurate DNA/MO injection. Simple adjustments can be made to the electroporation chamber to suit the shape of different aged embryos and to alter the size and location of the targeted region. This procedure can be used to electroporate separate regions of the CNS in the same embryo allowing separate manipulation of growing axons and their intermediate and final targets in the brain. CONCLUSION: Our study demonstrates that electroporation can be used as a versatile tool to investigate molecular pathways involved in axon extension during Xenopus embryogenesis. Electroporation enables gain or loss of function studies to be performed with easy monitoring of electroporated cells. Double-targeted transfection provides a unique opportunity to monitor axon-target interaction in vivo. Finally, electroporated embryos represent a valuable source of MO-loaded or DNA transfected cells for in vitro analysis. The technique has broad applications as it can be tailored easily to other developing organ systems and to other organisms by making simple adjustments to the electroporation chamber.

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Species referenced: Xenopus laevis

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	Figure 1. Efficient DNA transfection of stage 26–28 Xenopus embryos. a: Schematic representation of the experimental setup. Embryos were placed in the main channel of the electroporation chamber, while the electrode tips (0.5 mm wide) were positioned in the transverse channel. A diagram of the setup is presented as an insert with channel (outlines in red). b, c: Representative images of embryos electroporated in 1× MMR and 0.1× MBS. Bright field images (left panel) and GFP fluorescence (right panel) of living embryos 12 h after electroporation. No morphological abnormalities are observed. d: Histograms presenting the relative transfection efficiencies (blue) evaluated from observation of embryos as shown in c and d. The percentage of embryos showing macroscopic damage (red) was recorded for each condition. Different parameters are listed in the following order: Voltage, pulse duration, interpulse space and number of pulses. e, f: Electroporation resulted in a high percentage of transfected cells without affecting brain microanatomy. Nls-GFP signal (e) was observed in many nuclei (f) from the ventricle to the most superficial layer 48 h after electroporation. The transfected hemi-brain was outlined in white. Scale bars: 400 μm in b and c; 100 μm in e.
	Figure 2. Cell types and morphology of the transfected cells. a: Membrane-tethered GFP (GAP-GFP) delineated the processes of transfected neurons including the axons (the ventricle and neuropil are outlined in white). The arrow indicates a bundle of axons travelling in the neuropil). b: Radial-glia like morphology of GAP-RFP transfected cells lining the ventricle. c-e: Co-expression of GAP-GFP (c) and acetylated-tubulin (d) in superficial layers (e- merge). f: Wholemount brain preparation from an electroporated embryo showing different axon tracts. The brain outline was drawn based on the corresponding bright field image. Di., diencephalon; OT, optic tectum; Tel., telencephalon; Epi., epiphysis. Scale bars: 100 μm in f; 50 μm in a; 10 μm in b-e.
	Figure 3. Electroporation of stage 21–35/36 embryos leads to rapid expression of transgenes. a: Electroporation efficiency decreased with increasing embryonic stage. Percentages of nls-GFP positive cells 12 h after transfection at stage 26, 28 or 32 (n represents the number of sections analyzed from 3 embryos). Similar results were obtained at 48 h post electroporation (data not shown). b-d: Distribution of transfected cells depended on the stage of embryos electroporated. Distribution of nls-GFP transfected cells 48 h afterwards in embryos electroporated at stage 28 (b) and 32 (c). Note that the density of cells (DAPI) is lower laterally. d: Histograms showing decreases in the fraction of cells transfected in the superficial third of the brain when embryos were electroporated at stage 32 as compared to stage 28. e: A cluster of superficially located cells can be selectively transfected by injecting the DNA solution under the skin (the pia and epidermis are outlined in white). f-h: Time course of GFP expression in embryos electroporated at stage 29/30 (20 V/25 ms/1 s/8 x). The fractions as well as mean intensities of GFP positive cells were quantified (h) from sections (examples: f and g) (15 sections from 3 embryos were analyzed for the 6 h and 48 h time points and 39 sections from 3 embryos for the 24 h time-point). Differences between the time points were statistically significant using a Mann-Whitney test; probabilities are indicated together with the standard error (S.E.M). Outlines of the brains are presented (ventricle on the left). Scale bars: 100 μm in e; 50 μm in b, c, f and g.
	Figure 4. Using electroporation to study retino-tectal projections in vivo: a-b: Regions of the brain can be differentially targeted by sliding the embryo in the main channel (compare upper and lower panels in a). When the caudal part of the head was exposed, most of the optic pathway was electroporated (b). c-e: The transfected area can be restricted by reducing the amount of embryo area directly facing the electrodes. The modified chamber used to restrict electroporation is depicted in c (note the narrowing of the transverse channel in the inset), and a representative example of GFP expression 12 h post electroporation in a live embryo is shown in d. GFP expression in the tectum is shown on a wholemount dissected brain (e). Axons emanating from these neurons can be clearly observed (arrow). The dashed line delineates the diencephalon/mesencephalon boundary. The transfected area is restricted to the OT (dorsal mesencephalon). f-g: Electrodes can be placed dorsal and ventral to the embryo to target the ventral or dorsal part of the brain. A frontal section through the midbrain (g) demonstrating that ventral populations can be targeted by placing the embryo on its side in the specifically designed chamber represented in f. h-r: Retinas can be electroporated without affecting eye development. 48 h post electroporation, GAP-GFP was detected in all the retinal layers and outlined different retinal cell types with their characteristic morphologies (h-i). Eye microanatomy appeared normal (h). Eye-targeted electroporation can be performed by placing the embryo ventral side up, so that the eye but not the brain faces the electrodes (j). Eye-specific electroporation can be performed with limited brain transfection. Insert: side view of a transfected embryo 24 h after eye-targeted electroporation. GFP signal was detected in the eye and the RGC axons navigating to the tectum (arrow) but not in the brain on frontal sections (k). l-n: Co-electroporation of pCS2GAP-RFP with pEGFP. Most of the GAP-RFP positive cells (m) are also EGFP positive (n). Double positive cells are marked with white dots and the arrows point to axons leaving the retina. Outlines of the retina and lens were drawn from the corresponding DAPI counterstainings. After GAP-GFP electroporation, axons can be monitored using time-lapse microscopy (o-q) and growth cone morphology can be analyzed (r) in wholemount brain preparations. Axons were monitored as they entered the tectum. Initial positions of the two growth cones are indicated (white dot and rectangle). Time is in hours. Epi., epiphysis. Scale bars: 400 μm in a, d and insert j; 200 μm in b and e; 100 μm in k; 50 μm in h, i and l; 25 μm in o-q; 10 μm in m and n; 5 μm in r.
	Figure 5. Both retinal projection neurons and their substrate pathway can be manipulated separately in the same embryo. a-d: Eye-targeted electroporation can be combined with brain electroporation. a: A dorsal view of an embryo doubly transfected. Retinal axons (red in b and c) navigate normally to the tectum, passing through a transfected region of the diencephalon (green in c) (dashed line indicates the OT boundary). Eye- and ventral-targeted electroporation can be combined (d). Frontal section showing axons from the transfected retina (red) that have crossed the transfected midline (GFP-transfected) and growing dorsally towards tectum (arrow). e-g: Electroporation can be performed on embryos lipofected in the eyes. e: High magnification of two GFP lipofected axons passing through a cluster of electroporated tectal cells. f and g: Frontal sections of an embryo lipofected in the eye and electroporated in the brain. Retinal axons in the dorsal brain (green: f, g) traversed the transfected cells (red: g). Outlines of brains in wholemounts (b, c, e) and sections (f, g) were drawn based on bright field images and DAPI counterstainings respectively. Epi., epiphysis; Di., diencephalon; OT, optic tectum; Tel, telencephalon. Scale bars: 400 μm in a; 100 μm in b-g.
	Figure 6. Introducing Morpholinos into young Xenopus tadpoles by electroporation and in vitro approaches. a-d: Frontal sections of embryos 24 h after electroporation with lissamine-tagged MO. Large numbers of cells can be loaded with MO in both the brain (a) and the eye (c). Microanatomy of both structures appears normal (b and d). e-f: Co-electroporation of pCS2GAP-GFP with lissamine-tagged special delivery MO. e: A higher magnification image of a co-electroporated brain. The MO signal was de-saturated in Photshop in order to facilitate observation of MO and membrane GFP co-expression (arrowhead). f: An image of eye-targeted co-electroporation illustrating the extent of co-electroporation and the sizes of MO and DNA electroporated regions. g: Frontal section of a MO/GFP co-electroporated embryo showing that GFP can be used to trace the axons of electroporated cells (arrowheads indicate axons at different points in their pathway). h and i: Examples of embryos electroporated with pCS2GFP in the presence (i) or absence (h) of anti-GFP MO. Morphology of the eye appeared normal in both conditions (left panel). The GFP signal was sharply reduced in the anti-GFP MO condition when analyzed 12 h after electroporation (central panels). A decrease in electroporation efficiency was not a confounding factor in this experiment as the Special Delivery lissamine-tagged MO control is efficiently loaded in both conditions (far right panel). j: Quantification of results presented in h and i (n indicates the number of embryos analyzed). Anti-GFP MO only affects expression of pCS2GFP but not of pEGFP (Clontech). k: Anti-GFP MO was co-electroporated with GFP and GAP-RFP. 48 h after electroporation, GFP and RFP fluorescence was quantified on sections and the ratio between the two calculated. (n refers to the numbers of sections quantified [3 embryos were analyzed for control and 6 for MO]). Statistical analysis: Mann-Whitney test; probabilities are indicated together with the S.E.M. l-m: Sections through an eye lipofected with GFP (green, l and m) and subsequently loaded with lissamine-tagged MOs (red) using electroporation (merge, m). n-q: Electroporated embryos can be a source of modified cells for in vitro studies. Explants and cells cultured from MO (n and o) or DNA (GFP) (p and q) electroporated embryos. Scale bars: 400 μm in h; 100 μm in a; 50 μm in d, f, and g; in 25 μm e and m; 20 μm in n; 10 μm in o.

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