Dec 12, 2024
Lateral Tail Vein Bleeding for Blood Collection in Mice
- Jayshen Arudkumar1,
- Yu C.J. Chey1,
- Sandra Piltz1,
- Paul Quinton Thomas1,
- Fatwa Adikusuma1
- 1University of Adelaide
Protocol Citation: Jayshen Arudkumar, Yu C.J. Chey, Sandra Piltz, Paul Quinton Thomas, Fatwa Adikusuma 2024. Lateral Tail Vein Bleeding for Blood Collection in Mice. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp3kr8vzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 22, 2024
Last Modified: December 12, 2024
Protocol Integer ID: 93937
Keywords: Serum, Mouse, Creatine Kinase, Phenotyping, DMD
Disclaimer
These protocols are for research purposes only.
Abstract
Paper abstract: CRISPR-Cas9 gene-editing technology has revolutionised the creation of precise and permanent modifications to DNA, enabling the generation of diverse animal models for investigating potential treatments. Here, we provide a protocol for the use of CRISPR-Cas9 to create murine models of Duchenne Muscular Dystrophy (DMD) along with a step-by-step guide for their phenotypic and molecular characterisation. The experimental procedures include CRISPR microinjection of embryos, molecular testing at the DNA, RNA, and protein levels, forelimb grip strength testing, immunostaining and serum creatine kinase (CK) testing. We further provide suggestions for analysis and interpretation of the generated data, as well as the limitations of our approach. These protocols are designed for researchers who intend on generating and using mouse models to study DMD as well as those seeking a detailed framework of phenotyping to contribute to the broader landscape of genetic disorder investigations.
Protocol summary: Here we demonstrate the sampling of blood from the lateral tail vein of mice. Tail vein bleeding is necessary to isolate serum for downstream assessment of serum creatine kinase (CK) levels in our mouse model of Duchenne Muscular Dystrophy (DMD). We anticipate elevated serum CK levels in the DMD model compared to its wildtype counterparts, serving as a key indicator of the pathological changes associated with DMD.
Image Attribution
BioRender was used to generate figures for this manuscript.
Materials
- Individually ventilated cages (IVC)
- PPE (Personal Protective Equipment) - gloves, scrubs, mop cap, and unit shoes
- Empty IVC cage with bedding
- 70% ethanol, F10 SC Veterinary Disinfectant
- Scalpel blade
- Non-treated Microvette‱ CB300 Blood collection System (Kent Scientific)
- EMLA cream (5%)
- Tissues
- Eppendorf 1.5 or 2 ml tubes
- Box (or Esky) and ice
- Marker pen
- Small gauze or swaddle
Safety warnings
Wear proper PPE (gloves, safety goggles, enclosed shoes and lab coat) and prepare solvents in a chemical fume hood. Dispose used solvents or waste material in an appropriate biohazard waste containers.
Ethics statement
Animal work described in this manuscript has been approved and conducted under the oversight of the Animal Ethics Committee of South Australian Health and Medical Research Institute (SAHMRI) and The University of Adelaide.
Before start
Ensure there is suitable approval from your local Animal Ethics Committee for the maximum allowable blood volume to be taken per mice and that any operator is properly trained by their institution prior to commencing this protocol.
Preparation
Preparation
Label microvette tubes with animal ID
Prepare ice and transport facilities
Arrange microvettes for easy access during bleeding
Prepare the empty IVC cage with bedding
Skin Numbing and Thermacage Preparation
Skin Numbing and Thermacage Preparation
Take the cage of animals and check to see if each mice present no health concerns.
Apply EMLA cream to the tail's incision area and leave mice to sit for 5-min.
Use Thermacage heat box and ensure it is warmed to 37°C.
Place the animal in the Thermacage and let them sit for no longer than 10-min.
Gently place the animal in the restrainer – holding on to its tail.
Tail Bleeding and Collection
Tail Bleeding and Collection
Make a small incision over the lateral tail vein.
Encourage blood flow and collect into the Microvette tube.
Stop bleeding by applying gentle pressure using gauze and return animal to the empty IVC cage to sit for 5-min
If vein stops bleeding early, nick opposite vein if more blood required to be collected
Return the animal to its home cage and keep samples at room temperature
Serum Isolation
Serum Isolation
Ensure blood samples are left for 15-30-min from their last collection
Spin samples at 2000 RCF for 10-min at 4°C
Take colourless serum off the blood clot and place in microcentrifuge tube.
Freeze serum in -80°C.