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

Perfusão sanguínea em rãs africanas com garras para transcriptômica e proteômica

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.

Autor em destaque: Perfusão sanguínea rápida e eficaz em Xenopus  

Perfusão sanguínea rápida eficaz em Xenopus

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

Preparation for Perfusion in Xenopus   

Preparação para Perfusão em Xenopus

Após 15 minutos de submersão na solução de eutanásia, certifique-se de que o Xenopus está anestesiado beliscando o pé da rã. Registre o peso e quaisquer outros parâmetros necessários neste momento. Usando uma agulha calibre 31, injetar Xenopus laevis em cada músculo do membro torácico com 250 microlitros de heparina em PBS.injetar Xenopus tropicalis com 100 microlitros de heparina em PBS. Corte a ponta de uma agulha de perfusão com cortadores de fio. Conecte a agulha aparada à bomba e circule 54 unidades por mililitro de perfusato PBS heparinizado.Limpe todas as bolhas de ar da tubulação. Coloque a bandeja de dissecção em uma inclinação dentro de um recipiente secundário para facilitar a drenagem do sangue.

Research

JoVE Journal

Biology

Preparation for Perfusion in Xenopus

191 visualizações. Após 15 minutos de submersão na solução de eutanásia, certifique-se de que o Xenopus está anestesiado beliscando o pé da rã. Registre o peso e quaisquer outros parâmetros necessários neste momento. Usando uma agulha calibre 31, injetar Xenopus laevis em cada músculo do membro torácico com 250 microlitros de heparina em PBS.injetar Xenopus tropicalis com 100 microlitros de heparina em PBS. Corte a ponta de uma agulha de perfusão com cortadores de fio. Conecte a agulha apara...

Vídeo: Preparation for Perfusion in Xenopus   

Effective Rapid Blood Perfusion in Xenopus

Xenopus have been powerful model organisms for understanding vertebrate development and disease for over 100 years. Here, a rapid blood perfusion protocol in Xenopus, aimed at a consistent and drastic reduction of blood within all tissues, is defined. Perfusion is carried out by inserting a needle directly into the ventricle of the heart and pumping heparinized phosphate-buffered saline (PBS) through the vascular system. The procedure can be completed in approximately 10 min per animal. The blood is dominated by a few highly abundant proteins and cell types, creating numerous issues as these proteins mask most other molecules and cell types of interest. The reproducible characterization of adult Xenopus tissues with quantitative proteomics and single-cell transcriptomics will benefit from applying this protocol prior to organ sampling. The protocols for tissue sampling are defined in companion papers. These procedures are aimed at the standardization of practices across Xenopus of different sex, age, and health status, specifically X. laevis and X. tropicalis.

Presented here is an effective rapid blood perfusion protocol to prepare tissue samples from African clawed frogs for transcriptomics and proteomics studies.

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