Name: Preparation for Perfusion in Xenopus
Uploaded: 2023-05-16T12:50:00
Description: Watch this Scientific Journal Video about Preparation for Perfusion in Xenopus at JoVE.com

The video outlines a protocol for euthanizing and preparing Xenopus frogs, detailing the necessary anesthetic procedures and injections with heparin. It emphasizes proper techniques for blood drainage and equipment setup to ensure a humane and efficient process for handling these amphibians in a laboratory setting.

preparation for perfusion in xenopus

Blood Perfusion in African Clawed Frogs for Transcriptomics and Proteomics

The whole body perfusion of amphibians is routinely completed for the purposes of preservation and fixation1,2,3,4,5,6. However, these procedures occur at a rate that limits the number of fresh samples that can be taken per animal. The goal of this work is to develop an effective blood perfusion protocol in Xenopus, prioritizing the speed of the technique. The protocol takes less than 10 min per animal for X. tropicalis and less than 15 min per X. laevis animal. The secondary priorities are the ease of replication and the use of easily acquired equipment so that high-quality samples can be shared widely between Xenopus labs.
Xenopus frogs are used widely in biomedical research to study fundamental biological and pathological processes conserved across species. This tetrapod has a closer evolutionary relationship with mammals than other aquatic models, having lungs, a three-chambered heart, and limbs with digits. The international community effectively uses Xenopus to gain a deeper understanding of human disease through in-depth disease modeling and molecular analysis of disease-related gene function. The numerous advantages of Xenopus as an animal model make them invaluable tools to study the molecular basis of human development and disease; these advantages include: large oocyte and embryo size, high fecundity, ease of housing, rapid external development, and ease of genomic manipulation. Xenopus have been estimated to share ~80% of the identified human disease genes7.
Compared to popular mammalian models, Xenopus is a rapid, cost-effective model, with the ease of morpholino knock-down and availability of efficient transgenics and targeted gene mutations using CRISPR8. Quantitative mass spectrometry and single-cell transcriptomics have been successfully applied to Xenopus embryos9,10,&#160;but a recent cell atlas from Xenopus laevis shows that the composition of most tissues is dominated by blood cell types11. By developing a technique that exsanguinates tissue at a rapid rate and using chilled media, the sample freshness is minimally affected by perfusion. This is particularly important for applications where the goal is to profile physiologically unperturbed mRNA or protein expression.

Author Spotlight: Effective Rapid Blood Perfusion in Xenopus  

Effective Rapid Blood Perfusion in Xenopus

This procedure centers around the humane utilization of amphibian models in a manner that maximizes efficiency. Standardization practices can be applied to samples taken and preserved at the originating colony, mitigating biosecurity risks and eliminating animal transport stress. Profusion can be completed quickly, maximizing the time available to sample tissues while they are still fresh.This freshness is especially important for proteomic and transcriptomic profiling. This protocol is detailed and explicit, making it usable for individuals with a wide variety of backgrounds. This facilitates the sharing of high-quality samples between Xenopus labs.Along with this profusion technique, a similarly comprehensive protocol is being worked on for the standardized sampling of all major organs.

Watch this Scientific Journal Video about Preparation for Perfusion in Xenopus at JoVE.com

Preparation for Perfusion in Xenopus   

Preparation for Perfusion in Xenopus

Following 15 minutes of submersion in the euthanasia solution, ensure the Xenopus is anesthetized by pinching the foot of the frog. Record the weight and any other parameters needed at this time. Using a 31 gauge needle, inject Xenopus laevis in each forelimb muscle with 250 microliters of heparin in PBS.inject Xenopus tropicalis with 100 microliters of heparin in PBS. Trim the tip off a perfusion needle with wire cutters. Attach the trimmed needle to the pump and circulate 54 units per milliliter of heparinized PBS perfusate.Purge all air bubbles from the tubing. Place the dissection tray at an incline within a secondary container to facilitate blood drainage.

Research

JoVE Journal

Biology

Xenopus have been powerful model organisms for understanding vertebrate development and disease for over 100 years. Here, a rapid blood perfusion protocol ...