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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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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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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.

Mentions: Lissamine-tagged MOs were electroporated into both the brains and the eyes of stage 22 to 33/34 embryos with a success rate of over 80% with all four settings used (20 V/50 ms/1 s/8 x, 18 V/50 ms/1 s/10 x, 18 V/25 ms/1 s/10 × or 15 V/50 ms/1 s/10 x). In transverse sections, MO-loaded cells were evenly distributed throughout the width of the electroporated side of neuroepithelium, and along most of its dorso-ventral axis (Figure 6a and 6b). With eye-targeted electroporation, MO-positive cells were found in all of the cellular layers of the retina (Figure 6c and 6d). In all conditions tested, MO fluorescence was still detectable 48 h 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)

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

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Show All Figures
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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.
Mentions: Lissamine-tagged MOs were electroporated into both the brains and the eyes of stage 22 to 33/34 embryos with a success rate of over 80% with all four settings used (20 V/50 ms/1 s/8 x, 18 V/50 ms/1 s/10 x, 18 V/25 ms/1 s/10 × or 15 V/50 ms/1 s/10 x). In transverse sections, MO-loaded cells were evenly distributed throughout the width of the electroporated side of neuroepithelium, and along most of its dorso-ventral axis (Figure 6a and 6b). With eye-targeted electroporation, MO-positive cells were found in all of the cellular layers of the retina (Figure 6c and 6d). In all conditions tested, MO fluorescence was still detectable 48 h 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