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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 - BMC Dev. Biol. (2007)

Bottom Line: Blastomere injection of mRNA or antisense oligonucleotides has proven effective in analyzing early gene function in Xenopus.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK. jf348@cam.ac.uk

ABSTRACT

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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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.
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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.

Mentions: Embryos were electroporated following intraventricular pCS2GFP-DNA injection at stages ranging from 21 to 35/36 in chambers specially adapted to their morphology. External inspection of embryos under the fluorescent strereomicroscope showed that the fraction of embryos exhibiting bright GFP expression 12 h after electroporation was >70%, regardless of the stage at which the electroporation was performed. Despite these similar levels at a gross level, analysis of the GFP positive cell fraction (nls-GFP) on transverse sections revealed that a sharp decrease in efficiency occurs at stage 32 (Figure 3a). In addition, the GFP-expressing cells from late (stage 32) electroporations were distributed unevenly in the neuroepithelium. First, there was a marked decline in the number of transfected cells in the ventral brain. Indeed, the dorsal shift of the GFP-expression center of mass (relative to the DAPI nuclear marker) significantly increased 1.5-fold between stages 28 and 32. Secondly, 48 h after electroporation, fewer GFP-positive cells can be found in the superficial region of the neuroepithelium closest to the pia (Figure 3b–d). Several factors may contribute to the observed changes. As the brain develops, the ventricle lumen expands and post-mitotic cells migrate away from the ventricular surface to differentiate in superficial layers. Consequently, many cells are distant to the injection site, making them less likely to be transfected. In addition, a larger ventricle means a lower intraventricular concentration of injected DNA, which will restrict the transfection to cells lining the ventricle and decrease the electroporation efficiency overall. In agreement with this, doubling the injection volume enhances the electroporation success rate by 1.25 (assessed as in figure 1b; n = 12). However, if the local DNA concentration was the only factor involved, a stage-dependent distribution of GFP positive cells would be expected shortly after electroporation. In fact, 12 h after electroporation the fraction of GFP positive cells located in the superficial half of the brain was similar in embryos electroporated at stage 28 and 32 (24.4 ± 1.6, n = 36 and 21.9 ± 2.1, n = 20 sections respectively). The delayed onset of the stage-dependent difference in deep-superficial distribution suggests that it results at least partly from developmental changes in patterns of cell proliferation and migration that occur after electroporation.


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 - BMC Dev. Biol. (2007)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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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.
Mentions: Embryos were electroporated following intraventricular pCS2GFP-DNA injection at stages ranging from 21 to 35/36 in chambers specially adapted to their morphology. External inspection of embryos under the fluorescent strereomicroscope showed that the fraction of embryos exhibiting bright GFP expression 12 h after electroporation was >70%, regardless of the stage at which the electroporation was performed. Despite these similar levels at a gross level, analysis of the GFP positive cell fraction (nls-GFP) on transverse sections revealed that a sharp decrease in efficiency occurs at stage 32 (Figure 3a). In addition, the GFP-expressing cells from late (stage 32) electroporations were distributed unevenly in the neuroepithelium. First, there was a marked decline in the number of transfected cells in the ventral brain. Indeed, the dorsal shift of the GFP-expression center of mass (relative to the DAPI nuclear marker) significantly increased 1.5-fold between stages 28 and 32. Secondly, 48 h after electroporation, fewer GFP-positive cells can be found in the superficial region of the neuroepithelium closest to the pia (Figure 3b–d). Several factors may contribute to the observed changes. As the brain develops, the ventricle lumen expands and post-mitotic cells migrate away from the ventricular surface to differentiate in superficial layers. Consequently, many cells are distant to the injection site, making them less likely to be transfected. In addition, a larger ventricle means a lower intraventricular concentration of injected DNA, which will restrict the transfection to cells lining the ventricle and decrease the electroporation efficiency overall. In agreement with this, doubling the injection volume enhances the electroporation success rate by 1.25 (assessed as in figure 1b; n = 12). However, if the local DNA concentration was the only factor involved, a stage-dependent distribution of GFP positive cells would be expected shortly after electroporation. In fact, 12 h after electroporation the fraction of GFP positive cells located in the superficial half of the brain was similar in embryos electroporated at stage 28 and 32 (24.4 ± 1.6, n = 36 and 21.9 ± 2.1, n = 20 sections respectively). The delayed onset of the stage-dependent difference in deep-superficial distribution suggests that it results at least partly from developmental changes in patterns of cell proliferation and migration that occur after electroporation.

Bottom Line: Blastomere injection of mRNA or antisense oligonucleotides has proven effective in analyzing early gene function in Xenopus.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK. jf348@cam.ac.uk

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus