Plataforma de Vertebrados Acuáticos

1.   INTRODUCTION
2.   EQUIPMENT
3.   SERVICES
4.   CONTACT
5. REFERENCES
6. LINKS OF INTEREST

AQUATIC VERTEBRATES PLATFORM

The CABD-UPO Aquatic Vertebrate Platform was created on 2007, with funding from a Consolider-Ingenio Project (2007-2013) and under the management of Dr. José Luis Gómez-Skarmeta.

The Platform has been devoted ever since to the study of developmental mechanisms in lower vertebrates, within a technological environment that is in continuous expansion. Due to its complexity and size, this facility is unique in Europe and has now enabled 30 Spanish and international scientists to develop part of their research projects at the CABD.

2. EQUIPMENT

The main room of the facility houses:
- 23 multi-level racks, with 8 shelves each, adapted for 3-liter tanks; and
- 19 multi-level glass racks of 5 shelves, used for both zebrafish and medaka.
Additionally, there are 2 multi-level racks of 3 shelves for Xenopus laevis and 1 multi-level rack of 8 for Xenopus tropicalis.

The facility has three microinjection/transplantation stations available, each of them includes a stereo-microscope, a manipulator, and a high-precision injection pump.

3. SERVICES

The Aquatic Vertebrate Platform (supported by Junta de Andalucía) aims to facilitate research in aquatic vertebrate models for both national and international researchers as well as private companies. To this end, the scientific manager of the facility, Ana Fernández-Miñán, offers advice on genomic experimentation with zebrafish, medaka and Xenopus.

Specifically, we offer training, advice and the possibility to collaborate using the following techniques available at the Platform*:

A. GENERAL TRANSGENESIS

To build expression vectors à la carte we use:

A.1. The Tol2kit based on the multisite Gateway technology (1).

A.2. pTransgenesis vectors: compatible with Xenopus, zebrafish and medaka (2).

In addition to keeping a detailed record of the generated zebrafish transgenic lines, the Platform will also be in charge of their maintenance and distribution to interested researchers. (A web page with the available transgenic lines is under construction).

B. EMBRYOLOGY AND IMAGING

For the experimental testing of your transgenic lines, the following techniques are available:

B.1. Gain and loss-of-function studies by mRNA and morpholinos microinjection.

Morpholinos design and order information can be found at GENE TOOLS

B.2. Tailored protocols for RNA in situ hybridization and inmunohistochemistry.

B.3. Time-lapse in vivo confocal microscopy.

In collaboration with the Microscopy Facility at CABD (see rates here).

B.4. Mosaic analysis by transplantation and mosaic injection.

C. GENOMICS

We also offer a range of options for genomic analysis:

C.1. Enhancer activity assessment using the Zebrafish Enhancer Detector (ZED) vector (4).

The ZED vector consists of:

a) GFP under the control of a minimal promoter;
b) Insulator sequences to shield from positional effects;
c) Positive control for transgenesis (Cardiac Actin RFP).

C.2. Analysis of the insulating capacity of genetic elements.

We use an efficient vector designed to test an insulator’s functionality in zebrafish F0 injected embryos (4).

Insulator’s activity is shown by the ability of the element to downregulate the strong Z48 brain enhancer from the cardiac actin promoter.

C.3. Chromosome Conformation Capture (3C) technology (5).

The 3C-qPCR technique confirms distant interactions between two genomic positions, i.e. an enhancer and a promoter

C.4. Chip-PCR and Chip-seq techniques (6).

Chromatin inmunoprecipitation using specific antibodies and high-throughput sequencing

C.5. Basic bioinformatic support on Chip-seq experiments.

Available tools:

MACS (v.1.3.3) algorithm (Peak calling tool)
PinkThing
Gene Ontology Browser
Galaxy

The Aquatic Vertebrate Platform also offers a Host Laboratory to our visiting scientists, where you can develop collaboration projects with the expert technical support of our scientific manager Ana Fernández-Miñán.

* A, B1 and B2 services are free of charge for Consolider members. Rates for the rest of the services or rates for non-Consolider members must be discussed individually.
Should you be interested in any of the Platform services or in a collaboration, please contact the scientific manager.

4. CONTACT

Ana Fernández-Miñán
e-mail: amfermin(at)upo(dot)es
Phone number: +34 954 977445

5. REFERENCES

1. The Tol2kit: A Multisite Gateway-Based Construction Kit for Tol2 Transposon Transgenesis Constructs. Kwan et al. Developmental Dynamics 236:3088–3099, (2007).

2. pTransgenesis: a cross-species, modular transgenesis resource. Love, et al. Development 138, 5451-5458 (2011).

3. Transgenesis and gene trap methods in zebrafish by using the Tol2 transposable element. Kawakami K. Methods Cell Biol 77: 201-222, (2004).

4. Zebrafish Enhancer Detection (ZED) Vector: A New Tool to Facilitate Transgenesis and the Functional Analysis of cis-Regulatory Regions in Zebrafish. Bessa et al. Developmental Dynamics 236:3088–3099, (2007).

5. Quantitative analysis of chromosome conformation capture assays (3C-qPCR). H. Hage`ge et al. Vol.2 N0.7 Nature Protocols, (2007).

6. ChIP–seq: advantages and challenges of a maturing technology. Peter J. Park. Nature Reviews Genetics 10, 669-680 (2009).

6. LINKS OF INTEREST

1. Xenbase: Xenopus laevis and Xenopus tropicalis biology and genomics resource
www.xenbase.org

2. ZFIN: Zebrafish Model Organism Database
zfin.org

3. Zebrafish EST Database
www.lifesciences.sourcebioscience.com

4. Zebrafish Genome Resources
www.ncbi.nlm.nih.gov

5. MEPD: Medaka Expression Pattern Database
ani.embl.de

6. Medaka Genome Database
earth.lab.nig.ac.jp

7. USCS Genome Bioinformatics
genome.ucsc.edu

8. Ensembl
www.ensembl.org

Plataforma de Vertebrados Acuáticos

1. INTRODUCTION 2. EQUIPMENT 3. SERVICES 4. CONTACT 5. REFERENCES 6. LINKS OF INTEREST

AQUATIC VERTEBRATES PLATFORM

1. INTRODUCTION
2. EQUIPMENT
3. SERVICES
4. CONTACT
5. REFERENCES
6. LINKS OF INTEREST