Overview

Introduction

On the pages listed below you can find most common protocols used in the lab as well as a list of lab duties along with the names of the responsible persons. Please, contact the responsible persons if you have any question, suggestions or troubles.

Axolotl

Feeding

Artemia (Brine shrimp eggs)

Brine shrimp eggs are metabolically inactive and can remain in total stasis for two years while in dry oxygen-free conditions, even at temperatures below freezing. This characteristic is called cryptobiosis meaning "hidden life" (also called diapause). While in cryptobiosis, brine shrimp eggs can survive temperatures of liquid air (−190 °C or −310.0 °F) and a small percentage can survive above boiling temperature (105 °C or 221 °F) for up to two hours.

Once placed in brine (salt) water, the cyst-like eggs hatch within a few hours. The nauplii, or larvae, are less than 0.5 mm in length when they first hatch. Brine shrimp have a biological life cycle of one year, during which they grow to a mature length of around one centimeter on average. This short life span, along with other characteristics such as their ability to remain dormant for long periods, has made them invaluable in scientific research, including space experiments. This ability has also enabled the use of a hybrid of brine shrimp, bred to grow larger and live longer, as Sea-Monkeys.

Artemia Hatching Preparation: Protocol for 15 Ltr.

Switch on the bubbler.
Take the warm water from the tap and fill it into the container, meanwhile add 325 ml volume of salt (Red Sea Coral pro Salt), when the 15 Ltr. container is filled up the salt should be dissolved already.
Switch on the heating. And make sure that the light on the top is on ☺ Add 50-60 ml full of dried artemia (Stored at 40C) to this.

Hatching preparation

• The following protocol is considered for 15 l solution.
• Switch on the bubbler.
• Take the warm water from the tap and fill it into the container, meanwhile add 325 ml volume of salt (Red Sea Coral pro Salt), when the 15 Ltr. container is filled up the salt should be dissolved already.
• Switch on the heating. And make sure that the light on the top is on.
• Add 50-60 ml full of dried artemia (Stored at 40C) to this.

Keep it like this for 2 days with aeration. And >200 Lux of light.
Usually they hatch within few hours but It is good to keep for 2 days to achieve a good hatching.
During these two days temperature can be 18°-20°C.
Make sure hatching container is not getting clogged during these two days.

Lab duties

Chemicals

• Antibiotics
• Benzocaine
• Fibronectin
• Gelatine
• Liquid nitrogen
• Primary antibodies
• Secondary antibodies
• Serum immunostaining

Hardware

• Autoclave
• Cryostat
• Dissecting microscopes
• Leica confocal microscope
• Vacuum pumps
• Waterbath

General

• Ordering common stuff
• Heat-shock competent cells

Protocols

Cell culture

• Blastema cells
• Baculovirus titration
• Myoblasts electroporation
• Adhesive cell culture
• Electroporation of blastema cells
• Electroporation of neural epithelia cells (axolotl spinal cord)
• mESC culture/Cyst formation
• Neurosphere culture

Histology

Molecular biology

Booking calendars

Since some pieces of hardware are used intensively it is necessary to book them in advance. The links to the corresponding booking calendars are listed below.

• Fluorescence microscopes
  • Olympus SZX 16
  • New Olympus SZX 16
  • Olympus OVK
  • Axio Observer
• Microinjectors and electroporators
  • Electroporator
  • 2nd electroporator
  • Microinjector
  • Stereoscope
• Confocal microscopes
  • Leica LSM
• Cryostats
  • Cryostat 1
  • Cryostat 2
• Microtome
  • Microtome

Transgenic lines

Axolotl (A.mexicanum)

Name	Construct	Method of transgenesis	Author	Tissue	Image(s)	Remarks
PM28	Prrx1-TFP-NLS-T2A-mERt-Cre-ERt	Sce1	Prayag Murawala	Limb bud and limb blastema	Error creating thumbnail: File missing Limb bud expression	Germ-line transmitted
PM33	Col1A2-TFP-NLS-T2A-mERt-Cre-ERt	Sce1	Prayag Murawala	Bone and fibroblast	Error creating thumbnail: File missing Bone expression Error creating thumbnail: File missing Bone expression in limb bud	Germ-line transmitted

Digital data

Over the years the members of the lab have produced a lot of different data: Sanger sequences, EST libraries, images and so on. Select a category from the list below in order to view or download the data.

Theses

Title	Summary	Author
Reconstitution of three-dimensional neuroepithelia from mouse and human pluripotent stem cells		Yu Zhu

Databases

• Axolotl EST database contains several ESTs and also some additional information, e.g. the position of the well with the corresponding insert in the Blastema (BL) or Neural tube (NT) library.
• Axologle database contains several different Axolotl transciptome assemblies

Datasets

Sequences

Illumina

The data can be found under //biodata/groups/crtd_tanaka/axolotl/Sequencing/Illumina.

Name	Description	Tissue	Type	Read length	Reads	Format	Location	Author	Remarks
Mature spinal cord	Mature axolotl spinal cord	Spinal cord	Paired-ended	100bp	65,753,941	FastQ	SpinalCord/mSC.fastq.tar.gz	Akira Tazaki
Injured spinal cord	Injured axolotl spinal cord, 6dpi	Spinal cord	Paired-ended	100bp	78,459,950	FastQ	SpinalCord/6diSC.fastq.tar.gz	Akira Tazaki
Mature spinal cord	Mature axolotl spinal cord, replicate 2, crossing 142x5	Spinal cord	Paired-ended	100bp	tba	FastQ	SpinalCord/mSC_142x5.fastq.tar.gz	Akira Tazaki	Due to a sequencer failure only the first 66 bases are safe
Mature spinal cord	Mature axolotl spinal cord, replicate 3, crossing 96x6	Spinal cord	Paired-ended	100bp	tba	FastQ	SpinalCord/mSC_96x6.fastq.tar.gz	Akira Tazaki	Due to a sequencer failure only the first 66 bases are safe
Injured spinal cord	Injured axolotl spinal cord, 6dpi, replicate 2, crossing 142x5	Spinal cord	Paired-ended	100bp	tba	FastQ	SpinalCord/6diSC_142x5.fastq.tar.gz	Akira Tazaki	Due to a sequencer failure only the first 66 bases are safe
Injured spinal cord	Injured axolotl spinal cord, 6dpi, replicate 3, crossing 96x5	Spinal cord	Paired-ended	100bp	tba	FastQ	SpinalCord/6diSC_96x5.fastq.tar.gz	Akira Tazaki	Due to a sequencer failure only the first 66 bases are safe
Mature limb	Mature axolotl forelimb	Limb	Single-ended	76bp	65,934,228	FastQ	Limb/Mature.fastq.gz	Dunja Knapp
Amputated limb (36h)	Amputated axolotl forelimb (36hpa)	Limb	Single-ended	76bp	65,250,768	FastQ	Limb/36h_amp.fastq.gz	Dunja Knapp
Amputated limb (72h)	Amputated axolotl forelimb (72hpa)	Limb	Single-ended	76bp	62,970,898	FastQ	Limb/72h_amp.fastq.gz	Dunja Knapp
Lateral wound (36h)	Injured (lateral wound) axolotl forelimb (36hpw)	Limb	Single-ended	76bp	66,361,470	FastQ	Limb/36h_lw.fastq.gz	Dunja Knapp
Lateral wound (72h)	Injured (lateral wound) axolotl forelimb (72hpw)	Limb	Single-ended	76bp	64,133,834	FastQ	Limb/72h_lw.fastq.gz	Dunja Knapp

Sanger

The data can be found under //biodata/groups/crtd_tanaka/axolotl/Sequencing/Sanger.

Name	Description	Tissue	Sequences	Location	Author	Remarks
LimbBlastema	Limb blastema library	Limb blastema	20160 (RSD) / 15648 (SCF)	SCF files: LimbBlastema/LB_SCF.gz RSD files: LimbBlastema/LB_RSD.gz	Akira Tazaki
NeuraTube	Neural tube library	Teural tube	4896 (RSD) / 7905 (SCF)	SCF files: NeuralTube/NT_SCF.gz RSD files: NeuralTube/NT_RSD.gz	Akira Tazaki

Roche/454

The data can be found under //biodata/groups/crtd_tanaka/axolotl/Sequencing/454.

Name	Description	Tissue	Reads	Format	Location	Author	Remarks
454	Mixed 454	Mixed	1,208,954	Fasta/Qual	Ax_454.tar.gz	n/a