Bulk Electroporation-Mediated Gene Transfer intoXenopusTadpole Brain

Abstract

In vivo gene transfer is a powerful tool for investigating protein function and gene regulation in living organisms. Delivery of plasmid DNA to the brain of Xenopus tadpoles by bulk electroporationmediated (EM) gene transfer can be used to study the effects of ectopic gene expression on development, physiology, and behavior. It can also be used to mark cells for lineage tracing, investigate the in vivo function of gene regulatory elements when linked to a reporter gene, and introduce mutations into the genome of transfected cells, among other applications. Bilateral EM gene transfer allows for transfection of both sides of the brain, whereas unilateral EM gene transfer enables analysis of the effects of forced gene expression on one side of the brain, with the other side serving as the control. MATERIALSIt is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.RECIPES: Please see the end of this protocol for recipes indicated by <R>. Additional recipes can be found online at

Methods for Investigating the Larval Period and Metamorphosis in Xenopus

“…Studying gene function in mammals often involves genetically modified lines. However, Protocol: Bulk Electroporation-Mediated Gene Transfer into Xenopus Tadpole Brain Sáenz de Miera et al 2018) and Protocol: In Vivo Transfection of Naked DNA into Xenopus Tadpole Tail Muscle (Marshall et al 2017) open the door to rapid and targeted studies of gene function, thus bypassing the requirement for lengthy and laborious characterization of genetically modified lines. A central, contemporary question in gene regulation concerns chromatin conformation, transcription factor binding, and the location and role of enhancers in transcription initiation.…”

“…The focal biological levels queried by the protocols introduced here are indicated. For tissue harvest, see Protocol: Xenopus Tadpole Tissue Harvest (Patmann et al 2017); for transfection, see Protocol: Bulk Electroporation-Mediated Gene Transfer into Xenopus Tadpole Brain (Sáenz de Miera et al 2018) and Protocol: In Vivo Transfection of Naked DNA into Xenopus Tadpole Tail Muscle (Marshall et al 2017); for organ culture, see Protocol: Organ Culture of the Xenopus Tadpole Intestine (Ishizuya-Oka 2017); for cell culture, see Protocol: Cell Proliferation Analysis During Xenopus Metamorphosis: Using 5-Ethynyl-2-Deoxyuridine (EdU) to Stain Proliferating Intestinal Cells (Okada and Shi 2017); for ChIA-PET, see Protocol: Chromatin Immunoprecipitation for Chromatin Interaction Analysis Using Paired-End-Tag (ChIA-PET) Sequencing in Tadpole Tissues (Buisine et al 2018a) and Protocol: Chromatin Interaction Analysis Using Paired-End-Tag (ChIA-PET) Sequencing in Tadpole Tissues (Buisine et al 2018b).…”

Tissue-specific in vivo transformation of plasmid DNA in Neotropical tadpoles using electroporation

“…Among amphibians, most electroporation protocols were developed for model species, such as Xenopus, axolotls, and newts ( [14][15][16]; but see [17]). Indeed, there are many protocols detailing methods optimized for Xenopus tadpoles that include electroporation of brains [3,8,[18][19][20][21][22], eyes [23], and tails [16,24]. Here, we develop electroporation protocols for tadpoles of five species of Neotropical poison frogs (Dendrobatidae), cryptic forest/poison frogs (Aromobatidae), and glassfrogs (Centrolenidae).…”