The use of transgenics in the laboratory axolotl

Tilley, Lydia; Papadopoulos, Sofia-Christina; Pende, Marko; Fei, Ji-Feng; Murawala, Prayag

doi:10.1002/dvdy.357

Cited by 9 publications

(8 citation statements)

References 87 publications

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“…Reproductive characteristics of animal laboratory models used for transgenesis/genome editing experiments. References: (a) (Butt et al, 1992), (b) (Akazawa et al, 2021), (c) (Dominguez & Edwards, 2010), (d) (Shankland et al, 1992), (e) (Iyer et al, 2019), (f) (Williams & Jékely, 2016), (g) (A. H. L. Fischer et al, 2010), (h) (Seaver, 2016), (i) (Ebert, 2005), (j) (Yamaguchi & Yoshida, 2018), (k) (Schröder et al, 2008), (l) (Howe, 1962), (m) (Lawrence et al, 2012), (n) (Wittbrodt et al, 2002), (o) (Jeffery, 2020), (p) (Blackburn & Miller, 2019), (q) (Beck & Slack, 2001), (r) (Wolf & Hedrick, 1971), (s) (Tilley et al, 2022), (t) (Phifer-Rixey & Nachman, 2015), (u) (Weber & Olsson, 2008), (v) (Darling et al, 2005), (w) (Kaliszewicz, 2011), (x) (Massaro & Rocha, 2008), (y) (Frank et al, 2001), (z) (Lechable et al, 2020), (aa) (Iglesias et al, 2004), (bb) (Meneely et al, 2019) Table 2 provides an overview of current metazoan models that have been acclimated in laboratory settings for experiments involving genetics and reverse genetics. This list includes a mix of models that have been developed for a long time, some for more than a century, with well-established protocols and numerous publications utilizing these techniques (such as the house mouse, fruit fly, and Caenorhabditis elegans), as well as several other models where efforts to develop genetic approaches are still in their early stages.…”

Section: Comparison With Other Metazoan Modelsmentioning

confidence: 99%

Fast Cycling Culture of the Marine AnnelidPlatynereis dumerilii

Legras

Ghisleni

Soilihi

et al. 2023

Preprint

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Platynereis dumerilii, a marine annelid, is a model animal that has gained popularity in various fields such as developmental biology, biological rhythms, nervous system organization and physiology, behaviour, reproductive biology, and epigenetic regulation. The transparency of P. dumerilii tissues at all developmental stages makes it easy to perform live microscopic imaging of all cell types. In addition, the slow-evolving genome of P. dumerilii and its phylogenetic position as a representative of the vast branch of Lophotrochozoans add to its evolutionary significance. Although P. dumerilii is amenable to transgenesis and CRISPR-Cas9 knockouts, its relatively long and indefinite life cycle, as well as its semelparous reproduction have been hindrances to its adoption as a reverse genetics model. To overcome this limitation, an adapted culturing method has been developed allowing much faster life cycling, with median reproductive age at 15 weeks instead of 6-8 months using the traditional protocol. A low worm density in boxes and a strictly controlled feeding regime are important factors for the rapid growth and health of the worms. Moreover, a genetic selection for fast-reproducing individuals has been applied to isolate a "Fast Forward" strain that can be used for egg microinjection. This culture method has several advantages, such as being much more compact, not requiring air bubbling or an artificial moonlight regime for synchronized sexual maturation, and necessitating only limited water change. A full protocol for worm care and handling is provided.

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Section: Comparison With Other Metazoan Modelsmentioning

confidence: 99%

Fast Cycling Culture of the Marine AnnelidPlatynereis dumerilii

Legras

Ghisleni

Soilihi

et al. 2023

Preprint

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“…Compared to zebrafish, axolotls are larger(up to 30 cm in length), allowing serial blood collection, more complicated surgical operations, and the use of imaging techniques such as magnetic resonance imaging, echocardiography, and positron emission tomography [80]. Zebrafish are more well-studied genetically, but there are developed genomic databases of the axolotl, and several transgenetic axolotl models have been applied [84][85][86][87][88]. How the axolotl can regenerate complex structures has not yet been determined.…”

Section: Chemical Ablation Of β Cellsmentioning

confidence: 99%

A Novel Animal Model in Diabetes Research: Regeneration of β cells in the Axolotl Salamander?

Sørensen¹,

Dittrich²,

Lauridsen³

2023

Preprint

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Diabetes is a group of diseases characterized by loss of β cell mass and/or -function, resulting in hyperglycemia. Approx. 537 million people worldwide suffer from diabetes – a number which is expected to increase. Diabetes is primarily treated by exogenous insulin, which comes with the challenges of maintaining glycemic control to prevent ketoacidosis and severe complications. The need for a curative treatment has initiated the research in β cell regeneration. Several studies in mice have identified essential genes for β cell fate, which can be manipulated in other cells to induce generation of new β cells. Zebrafish, a regeneration-positive animal model, has shown several different sources of new β cells, including regeneration by self-replication, neogenesis by duct-associated progenitor cells, and transdifferentiation of other endocrine islet cells. The animal models used in this research area are either limited by their low regenerative ability (mice), or their small size and remoteness from humans (zebrafish). There is a need for new animal models of diabetes, in which the molecular pathways of endogenous regeneration can be studied. This study proposes the axolotl salamander (Ambystoma mexicanum) as a model for studying the regeneration of β cells. The axolotl has shown great regenerative capability, as they have proven capable of regenerating amputated limbs, and hearts with myocardial infarction, among other organs. This study aims to establish a diabetic axolotl model, investigate their regenerative ability in the pancreas, and examine the potential systemic effects of the induced disease. In a pilot study, five different protocols using STZ (streptozotocin) were tested, and the most optimal protocol was found. Furthermore, the glucose tolerance test was optimized to characterize the glycemic state of the animals. The effect of the treatment on blood glucose levels was measured to characterize the development and decline of the disease. The histological changes in the pancreas were examined. Moreover, the systemic effects of the STZ treatment were investigated in blood and urine. The study indicated that it is possible to induce diabetes in the axolotl, but variations between the animals should be minimized, or the sample size should be increased to conduct a satisfying experiment, as it was not possible to induce diabetes in all animals. Regeneration was not observed histologically, but a restoration of blood glucose levels was seen over the span of the experiment. Lastly, edema formation was observed in some of the STZ-treated animals, but the cause of edema remains undetermined.

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“…By constitutive or conditional overexpressing targeted genes, the roles of thrombospondin-4 and p16 INK4a were investigated in spinal cord and limb regeneration 12 , 13 . However, transgenesis-based conditional gene overexpression requires the generation of transgenic animals, which can be time-consuming and is limited by the lack of well-characterized tissue-specific promoters 14 , 15 . In terms of knockout, it has been reported that introducing TALEN or CRISPR/Cas9 into fertilized eggs could efficiently lead to constitutive knockout/knockdown of targeted genes in axolotls 16 , 17 .…”

Section: Introductionmentioning

confidence: 99%

Establishing an Efficient Electroporation-Based Method to Manipulate Target Gene Expression in the Axolotl Brain

Fu,

Peng,

Zeng

et al. 2023

Cell Transplant

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The tetrapod salamander species axolotl ( Ambystoma mexicanum) is capable of regenerating injured brain. For better understanding the mechanisms of brain regeneration, it is very necessary to establish a rapid and efficient gain-of-function and loss-of-function approaches to study gene function in the axolotl brain. Here, we establish and optimize an electroporation-based method to overexpress or knockout/knockdown target gene in ependymal glial cells (EGCs) in the axolotl telencephalon. By orientating the electrodes, we were able to achieve specific expression of EGFP in EGCs located in dorsal, ventral, medial, or lateral ventricular zones. We then studied the role of Cdc42 in brain regeneration by introducing Cdc42 into EGCs through electroporation, followed by brain injury. Our findings showed that overexpression of Cdc42 in EGCs did not significantly affect EGC proliferation and production of newly born neurons, but it disrupted their apical polarity, as indicated by the loss of the ZO-1 tight junction marker. This disruption led to a ventricular accumulation of newly born neurons, which are failed to migrate into the neuronal layer where they could mature, thus resulted in a delayed brain regeneration phenotype. Furthermore, when electroporating CAS9-gRNA protein complexes against TnC ( Tenascin-C) into EGCs of the brain, we achieved an efficient knockdown of TnC. In the electroporation-targeted area, TnC expression is dramatically reduced at both mRNA and protein levels. Overall, this study established a rapid and efficient electroporation-based gene manipulation approach allowing for investigation of gene function in the process of axolotl brain regeneration.

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The use of transgenics in the laboratory axolotl

Cited by 9 publications

References 87 publications

Fast Cycling Culture of the Marine AnnelidPlatynereis dumerilii

Fast Cycling Culture of the Marine AnnelidPlatynereis dumerilii

A Novel Animal Model in Diabetes Research: Regeneration of β cells in the Axolotl Salamander?

Establishing an Efficient Electroporation-Based Method to Manipulate Target Gene Expression in the Axolotl Brain

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