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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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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.
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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.

Mentions: To further characterize the efficiency of electroporation, nucleus-targeted GFP (nls-GFP) was transfected to quantify the fraction of GFP-expressing versus non-expressing cells on transverse brain sections counterstained with a nuclear stain (DAPI). 48 h after electroporation (20 V/50 ms/1 s/8 x), the average fraction of cells expressing GFP per section was 47.1 ± 2.5% in the transfected region (n = 47 sections 6 embryos; Figure 1e and 1f). Transfected cells were scattered along 50–70% of the dorso-ventral axis and throughout the whole neuroepithelium. At this stage, the brain comprises a proliferative region adjacent to the ventricle lumen (ventricular zone) surrounded by layers of migrating and differentiating neurons (mantle zone). 48 h post-electroporation, transfected cells were present in both regions. GFP-expressing cells residing in the superficial third of the brain, populated by differentiated neurons, represented 31.8 ± 1.5% of total labeled cells (n = 32). However, the fraction of transfected cells in the superficial half of the brain increased with pulse duration (see additional file 1 a & b). To check the morphology of transfected cells and further characterize their cell types, we transfected membrane-targeted GFP or RFP (GAP-GFP and -RFP). As expected, the GFP signal was found from the ventricle to the neuropil (Figure 2a). Cells lining the ventricle could be seen extending radial process towards the pia, typical of dividing cells (Figure 2b). Transfected neurons appeared to differentiate normally as they expressed the neuronal marker acetylated tubulin, and sent long processes into the neuropil (Figure 2c–e). Furthermore, several axon tracts could be recognized in a whole-mount view of the brain (Figure 2f).


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)

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

License
Show All Figures
getmorefigures.php?uid=PMC2147031&req=5

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.
Mentions: To further characterize the efficiency of electroporation, nucleus-targeted GFP (nls-GFP) was transfected to quantify the fraction of GFP-expressing versus non-expressing cells on transverse brain sections counterstained with a nuclear stain (DAPI). 48 h after electroporation (20 V/50 ms/1 s/8 x), the average fraction of cells expressing GFP per section was 47.1 ± 2.5% in the transfected region (n = 47 sections 6 embryos; Figure 1e and 1f). Transfected cells were scattered along 50–70% of the dorso-ventral axis and throughout the whole neuroepithelium. At this stage, the brain comprises a proliferative region adjacent to the ventricle lumen (ventricular zone) surrounded by layers of migrating and differentiating neurons (mantle zone). 48 h post-electroporation, transfected cells were present in both regions. GFP-expressing cells residing in the superficial third of the brain, populated by differentiated neurons, represented 31.8 ± 1.5% of total labeled cells (n = 32). However, the fraction of transfected cells in the superficial half of the brain increased with pulse duration (see additional file 1 a & b). To check the morphology of transfected cells and further characterize their cell types, we transfected membrane-targeted GFP or RFP (GAP-GFP and -RFP). As expected, the GFP signal was found from the ventricle to the neuropil (Figure 2a). Cells lining the ventricle could be seen extending radial process towards the pia, typical of dividing cells (Figure 2b). Transfected neurons appeared to differentiate normally as they expressed the neuronal marker acetylated tubulin, and sent long processes into the neuropil (Figure 2c–e). Furthermore, several axon tracts could be recognized in a whole-mount view of the brain (Figure 2f).

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