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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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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.
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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.

Mentions: Since only the region lying between the two electrodes is efficiently electroporated, different areas can be selectively electroporated by sliding the embryo forward or backward in the main channel to expose the rostral or caudal part of the head (Figure 4a and 4b). This configuration gives rise to large transfected areas, extending rostro-caudally over 568 ± 40.5 μm (n = 13). Taking advantage of the insulating property of Sylgard, the electroporated region can be restricted by narrowing the transverse channel (158 ± 17.6 × 89 ± 8.7 μm, n = 14), making specific electroporation of the embryonic tectum or diencephalon feasible (Figure 4c–e). In addition, the relative orientation of embryos to the electrodes can be changed to drive DNA towards different regions. Using modified chambers, the ventral-most regions of the brain, which are usually difficult to electroporate, can be targeted, allowing electroporation of the optic chiasm region (Figure 4f and 4g).


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)

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.
© Copyright Policy - open-access
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Show All Figures
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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.
Mentions: Since only the region lying between the two electrodes is efficiently electroporated, different areas can be selectively electroporated by sliding the embryo forward or backward in the main channel to expose the rostral or caudal part of the head (Figure 4a and 4b). This configuration gives rise to large transfected areas, extending rostro-caudally over 568 ± 40.5 μm (n = 13). Taking advantage of the insulating property of Sylgard, the electroporated region can be restricted by narrowing the transverse channel (158 ± 17.6 × 89 ± 8.7 μm, n = 14), making specific electroporation of the embryonic tectum or diencephalon feasible (Figure 4c–e). In addition, the relative orientation of embryos to the electrodes can be changed to drive DNA towards different regions. Using modified chambers, the ventral-most regions of the brain, which are usually difficult to electroporate, can be targeted, allowing electroporation of the optic chiasm region (Figure 4f and 4g).

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