ANIMAL MODELS CORE FACILITY (AMCF)

Members

Coordinator: Prof. Concetta Ambrosino
Molecular Biology: Nicola Antonino Russo
Cell Biology: Carla Reale, Filomena Russo
Embriology: Luca Roberto

 

Genetically modified mouse models are one of the most powerful tool in the scientific areas, such as cancer, drug-induced immunotoxicity and development biology. Genetically engineered mice are constructed to more precisely model human phenotypes and pathologies, giving researchers a better way to explore disease mechanisms and drug discovery. Nowadays it is possible remove, replace and insert any kind of sequence in the mouse genome. Biogem offers quality custom generation of a wide range of mouse models, using diverse techniques including homologous recombination in embryonic stem (ES) cells, CRISPR/Cas9 system and a variety of advanced technologies of molecular biology, enabling you to obtain the precise models you need.

Our services include:

  • Targeting strategy design.
  • Targeting vector construction.
  • CRISPR/Cas9 system strategy.
  • Electroporation of mouse embryonic stem (ES) cells.
  • Assessing ES cells for homologous recombination.
  • Generating chimeric mice by morula or blastocyst injection.
  • Assessing germline transmission.
  • Breeding chimeric mice to heterozygosity.

Our services can be ordered together or individually depending on the investigator's needs. Moreover, we can generate chimeras also performing microinjection of ES clones obtained by consortia such as KOMP and EUCOMM.

Types of mouse models:

  • Constitutive knock out

A Constitutive Knock Out will eliminate the function of the targeted gene in all cells throughout development and adulthood. Traditional knock-out involves deleting or disabling both copies of a specific gene. It is indicated for study of gene function, drug and therapeutic development and disease research.

  • Conditional knock-out

Conditional knock-out involves deleting or disabling a gene in only a particular organ, tissue, or cell type or only during a certain development stage.
      These system utilizes the Cre-lox system. This is a site-specific DNA recombination through the    
      interaction of the Cre recombinase enzyme and loxP sites in the DNA strand: a mouse bearing  
     the recombinase-specific sites is bred with a mouse expressing the recombinase. The tissue-
     specific expression of the recombinase allows the inactivation of the gene of interest only in the
     tissue where the recombinase is expressed. Conditional knock-outs provide a temporal and
     tissue-specific gene knock-out control while allowing the mouse model to mature under the
     normal operating role of the gene of interest.

  • Constitutive/Conditional Point mutation

This approach involves the introduction of one or more point mutations anywhere in the target gene, to analyse the effect of SNPs. This method can also be combined with conditional approaches,  providing a temporal and tissue-specific gene mutation.

  • Rosa26 knock-in

This approach, called "knock-in", is based on targeted insertion of the transgene in a well-characterized, transcriptionally active locus in the mouse genome, such as ROSA26 locus, with multiple advantages over the pro-nuclear injection approach.

  • Humanization

Humanization, based on Knock-in approach, allows for in vivo testing of compounds and antibodies against human proteins in the mouse. This is achieved by the replacement of the murine gene with the human counterpart, placing the gene in the same transcriptional context as the murine gene.

  • Reporter fusions

This targeting vector allows the introduction of a reporter gene to follow gene expression

  • Constitutive/Inducible Cre- or FLP-driver mouse strains

The conditional approaches based on the Cre/lox system need mouse strains expressing the recombinase in a tissue- and time-specific manner, to make this models it is necessary the generation of a mouse that express Cre recombinase (constitutive or inducible) under the control of a specific promoter.

 

Other services:

  • Drafting for Ministerial authorization.
  • Genotyping.
  • In vivo Cre or Flp modification.
  • Mouse model phenotypical characterization.
  • Mouse sample collection.
  • Sperm freezing cryopreservation.
  • In vivo sperm fertilization (IVF).
  • SPF mice riderivation.

Zebrafish Service

Members

Coordinator: Prof. Concetta Ambrosino
Zebrafish modelling: Nicola Antonino Russo, Carla Reale
Toxicology: Alfonsina Porciello
Zebrafish Embriology and Breeding: Filomena Russo, Luca Roberto, Alfonsina Porciello

The zebrafish (Danio rerio) is the premier non-mammalian vertebrate model organism. During the last few decades, a unique set of properties has made the zebrafish a popular vertebrate model in various field of biology, ranging from developmental biology, human disease studies to environmental toxicology. The zebrafish is an outstanding system for studying processes difficult or impossible to follow in other animals such as the mechanisms underlying organogenesis. Unlike mice, this model system is readily amenable to forward genetic mutagenesis approaches for the identification of new genes required for these key developmental processes and functions. In the areas of vertebrate development biology and functional genomics, the transparency of the zebrafish embryo during development has allowed researchers to track regulation of gene expression using fluorescent protein genes in real time in living animals. Consequently, the conversion of genetic and other biological information learned from the fish to humans has been faster than in other vertebrate systems.

The Zebrafish facility in Biogem is equipped with breeding systems and also with experimental toxicology  systems. The breeding systems have a continuous water recirculation with temperature (° C), conductivity (μS) and pH values constantly controlled in order to guarantee the best conditions of fish growth. The toxicology systems, unlike those of breeding, does not shows any continuous recycling of water. The values ​​of temperature (° C), conductivity (μS) and water pH are constantly checked and it is possible to program water changes at specific time intervals in order to optimize the concentration of testing molecules and drugs.

The Zebrafish Service provides expertise for transgenic lines and mutants generation and support for design and realization of toxicology studies with the zebrafish model.

Our services include:

  • Animal housing and care.
  • Breeding and maintenance.
  • Embryo production for genetic knockdown, transgenic and chemical genetic testing
  • Strain propagation, including genotype services
  • Zebrafish embryo microinjection.
  • Zebrafish transgenesis by CRISPR/Cas9 technology
  • Morpholino oligonucleotide-mediated antisense gene-specific knockdowns
  • mRNA and cDNA-mediated gene misexpression
  • Expression pattern analysis of genes of interest (RNA in situ hybridization)
  • Important and rearing of mutant and transgenic lines
  • Embryonic phenotyping
  • Reproductive phenotyping
  • Animals treatment for toxicology studies under long term conditions
  • Zebrafish Samples collections