User:The Axolotl Neuroscientist/sandbox

The first experiments that successfully developed transgenic amphibians into embryos began in the 1980s with Xenopus laevis. Later, germline transgenic axolotls in Ambystoma mexicanum were produced in 2006 using a technique called I-SceI-mediated transgenesis which utilizes the I-SceI endonuclease enzyme that can break DNA at specific sites and allow for foreign DNA to be inserted into the genome. Both Xenopus laevis and Ambystoma mexicanum are model organisms used to study regeneration. In addition, transgenic lines have been produced in other salamanders including the Japanese newt Pyrrhogaster and Pleurodeles watl. Genetically modified frogs, in particular, Xenopus laevis and Xenopus tropicalis, are used in development biology with new research methods allowing for the study of late development GM frogs can also be used as pollution sensors, especially for endocrine-disrupting chemicals.[152] Many lines of transgenic X. laevis are used to study immunology to address how bacteria and viruses cause infectious disease at the University of Rochester Medical Center’s X. laevis Research Resource for Immunobiology (XLRRI). Amphibians can also be used to study and validate regenerative signaling pathways such as the Wnt pathway. The wound-healing abilities of amphibians have many practical applications and can potentially provide a foundation for scar-free repair in human plastic surgery, such as treating the skin of burn patients. Amphibians like X. laevis are suitable for experimental embryology because they have large embryos that can be easily manipulated and observed during development. In experiments with axolotls, mutants with white pigmented skin are often used because their semi-transparent skin provides an efficient visualization and tracking method for fluorescently tagged proteins like GFP. Amphibians are not always ideal when it comes to the resources required to produce genetically modified animals; along with the one to two-year generation time, Xenopus laevis can be considered less than ideal for transgenic experiments because of its pseudotetraploid genome. Due to the same genes appearing in the genome multiple times, the chance of mutagenesis experiments working is lower. Current methods of freezing and thawing axolotl sperm render them nonfunctional, meaning transgenic lines must be maintained in a facility and this can get quite costly. Producing transgenic axolotls has many challenges due to their large genome size. Current methods of generating transgenic axolotls are limited to random integration of the transgene cassette into the genome, which can lead to uneven expression or silencing. Gene duplicates also complicate efforts to generate efficient gene knockouts. Despite the costs, axolotls have unique regenerative abilities and ultimately provide useful information in understanding tissue regeneration because they can regenerate their limbs, spinal cord, skin, heart, lungs, and other organs. Naturally occurring mutant axolotls like the white strain that are often used in research have a transcriptional mutation at the Edn3 gene locus. Unlike other model organisms, the first fluorescently labeled cells in axolotls were differentiated muscle cells instead of embryos. In these initial experiments in the early 2000s, scientists were able to visualize muscle cell regeneration in the axolotl tail using a microinjecting technique, but cells could not be traced for the entire course of regeneration due to too harsh conditions that caused early cell death in labeled cells. Though the process of producing transgenic axolotls was a challenge, scientists were able to label cells for longer durations using a plasmid transfection technique, which involves injecting DNA into cells using an electrical pulse in a process called electroporation. Transfecting axolotl cells is thought to be more difficult because of the composition of the extracellular matrix (ECM). This technique allows spinal cord cells to be labeled and is very important in studying limb regeneration in many other cells; it has been used to study the role of the immune system in regeneration. Using gene knockout approaches, scientists can target specific regions of DNA using techniques like CRISPR/Cas9 to understand the function of certain genes based on the absence of the gene of interest. For example, gene knockouts of the Sox2 gene confirm this region’s role in neural stem cell amplification in the axolotl. The technology to do more complex conditional gene knockouts, or conditional knockouts that give the scientist spatiotemporal control of the gene is not yet suitable for axolotls. However, research in this field continues to develop and is made easier by recent sequencing of the genome and resources created for scientists, including data portals that contain axolotl genome and transcriptome reference assemblies to identify orthologs.