Genetically modified animal and cell models

Gene targeting and experimental embryology

(Dr. Nicola Russo and Dr. Luca Roberto)

- Genetically Modified Cellular Models

Genetically modified cell models are crucial tools in scientific research, used to better understand the functions of genes and their role in human diseases. These models are created by specifically altering the DNA of cells through techniques such as CRISPR/Cas9, gene targeting or viral vector transfection. Modifications may include the insertion, removal or mutation of genes, allowing researchers to analyze how these changes affect cellular processes, such as proliferation, differentiation and response to external stimuli. Genetically modified cell models are essential for studying complex diseases, developing new treatments and testing the efficacy of drugs in a controlled environment. These models offer a highly specific and reproducible system, helping to reduce the use of animal models and to promote a more ethical approach to biomedical research.

What the service does

Biogem offers a high quality custom generation of cell models using advanced molecular biology technologies.

List of services:

• Design of a custom strategy
• Construction of a targeted vector
• Electroporation, transfection, and infection of murine and human cell lines
• Selection, validation, and sequencing of inserted gene modifications

Types of cell models:

Cellular lines over-expressing a gene

Cellular lines over-expressing mutated genes

Knock-out Crispr/Cas9 cell lines 

Knock-in Crispr/Cas9 cell lines 

Reporter cell lines

Immortalized cell lines

- Genetically Modified Mouse Models

Genetically modified mouse models are essential tools for biomedical research, as they allow us to study the functions of specific genes and their role in human diseases. These models are generated by altering the DNA of mice through techniques such as gene targeting or genome editing exploiting the homologous recombination in embryonic stem (ES) cells. Genetic modifications may include the insertion, removal or mutation of specific genes, making possible for researchers to observe the biological effects of these alterations. These genetically modified mice are crucial for understanding the mechanisms of complex diseases such as cancer, neurodegenerative diseases and rare genetic diseases. They also serve as platforms for the development and testing of new drugs and gene therapies, accelerating the advancement of personalized medicine. Due to their importance, genetically modified mouse models became a standard in preclinical research and are a bridge between basic research and clinical applications.

What the service does

Biogem offers a high quality custom generation of mouse models using advanced molecular biology technologies.

List of services:

• Design of a custom strategy
• Writing of the project for ragulatory authorization
• Construction of a targeted vector
• Electroporation of mouse embryonic stem (ES) cells
• Evaluation of ES cells for homologous recombination
• Generation of chimeric mice by morula or blastocyst injection
• Import and use of ES clones obtained from consortia such as KOMP and EUCOMM
• Evaluation of germline transmission
• Breeding of chimeric mice by heterozygosity
• Import and breeding of genetically modified mice from international distributors
• Rederivation of acquired lines
• Sperm freezing of generated and acquired lines

Types of mouse models:

Constitutive knockout

A constitutive knockout mouse is a genetic model in which a specific gene is permanently deactivated in all cells of the organism, from birth to death. This model is used to study the essential functions of the gene in normal and pathological conditions.

Conditional Knockout

A conditional knockout mouse is a genetic model in which a specific gene can be turned off in a controlled manner, usually in a specific tissue or at a specific developmental stage, making possible a more precise study of gene function.

Constitutive/Conditional Point Mutation

A point mutation mouse is a genetic model in which a single DNA base has been modified to alter a specific gene. This makes possible to study the specific effects of that mutation, which is useful for understanding genetic diseases and for developing targeted therapies

Rosa26 Knock-in 

The Rosa26 knock-in mouse is a genetic model in which an exogenous gene is inserted into a specific locus (Rosa26) of the mouse genome. This site is commonly used to steadily and uniformly express transgenes, facilitating the study of exogenous or modified genes.

Humanized Mice

Humanization, based on the knock-in approach, allows in vivo testing of compounds and antibodies against human proteins in mice. This is achieved by replacing the murine gene with the human counterpart, inserting the gene into the same transcriptional context as the murine gene.

Reporter Mice

A reporter mouse is a genetic model in which a specific gene is replaced or associated with a visible marker, such as a fluorescent protein, to monitor gene expression or cellular activity in vivo.

Cre Mice

A Cre mouse is a genetically modified mouse model that expresses the enzyme Cre recombinase, which allows specific genes to be activated or deactivated in selected tissues or developmental stages. This system is used to study gene functions in a controlled and precise manner.

Zebrafish Facility 

(Dr. Carla Reale)

The zebrafish (Danio Rerio) is the main vertebrate organism model, not belonging to the class of mammals, whose use was initially relevant for the study of developmental biology and subsequently of various human diseases, including cancer, and for the evaluation of the toxicity of various molecules. Its success derives from the possibility of using it in the study of complex processes such as those related to organogenesis. In the field of oncology, the use of zebrafish larvae is currently widespread in order to monitor the invasive, metastatic, and proliferative capacity of human tumor cells (xenograft models).

What the service and its facility do

The Biogem Zebrafish facility is equipped with cutting-edge and technologically advanced breeding systems (Stand Alone system) that guarantee the maximum animal welfare, ensuring the maintenance of optimal aquatic parameters and the day/night cycle, as well as the appropriate technology for microscopic analysis of the larvae. The facility hosts several model lines generated for the study of different human pathologies and reporter lines that allow to visualize different cell populations in the animal through fluorescence from the early stages of life.

Biogem's zebrafish service mainly deals with:

• the generation of genetically modified in vivo models for the study of different human pathologies;
• the generation of xenograft models for the study of different types of tumor;
• the screening of molecules to evaluate their toxicological or antitumor effect.
• definition of the targeting strategy
• writing of the project for ragulatory authorization
• injection through transgenesis, constitutive and conditional knockouts, over-expression (CRISPR/Cas9 system)
• phenotypic characterization of the model
• behavioral studies
• sperm freezing of the lines generated
• in vitrofertilization (IVF)

In addition to generating genetically modified models for oncology research, we deal with the generation and analysis of xenograft models for the evaluation of the efficacy of different drugs in larvae and adults (for this purpose we have a fli1:GFP line that marks blood vessels and an immunosuppressed adult line):

• Cell Derived Xenograft (CDX) and Patient Derived Xenograft (PDX)
• high-resolution microscopic analysis (confocal and 2-photon microscope)
• analysis of the obtained data 

Furthermore, we provide support for in vivo high-throughput screening of molecules for:

• a rapid characterization of the effects in terms of the mechanisms of action and cytotoxicity
• determine the specific toxicity of molecules in reporter lines
• test the effect of anti-cancer therapies on tumor progression and metastatic invasion