Available cancer models are focused on different pharmacological aspects of tumor responses to new antitumor drugs. Our specific expertise is focused on in molecular and cellular biology, in vitro/vivo tests on murine and human xenograft models. More than 70 sub-cutaneous, orthotopic and metastatic xenograft models are available for the efficacy evaluation of new compounds. Our human tumor xenograft model portfolio includes cancer cell lines from the following tissues: brain, breast, central nervous system, colon, kidney, hematologic, lung, ovary, pancreas, prostate, skin and stomach.  All the experimental activities are performed under quality control by using GLP standard procedures.

Evaluation of tumors with quantitative bioluminescent imaging: The availability of a large panel of bioluminescent pre-clinical models allows the evaluation of tumor progression in real time using the optical molecular imaging system IVIS Spectrum from PerkinElmer.
Humanized Models: We have a large collection of primary tumors (biopsies derived from patients) xenografted in tumor-bearing mice (i.e. Lung cancers, Mesothelioma, and others).
Anti-cancer drug evaluation: Administration of therapeutic candidates through different routes (intravenous, subcutaneous, intraperitoneal, oral/gavage, intramuscolar, intratracheal, slow release devices) alone or in combination with traditional anti-tumoral drugs, with high standards of care.

Fertilization of a large number of oocytes (up to 200) using frozen or fresh sperm.
This procedure is particularly suited for the strong expansion of a colony with few males or to recover the colony whose males were infertile after repeated natural unsuccessful mating. The same protocol is used to thaw colonies cryopreserved by sperm freezing.

Glaucoma 
Intra Ocular Pressure (IOP) increase induces retina and ophthalmic nerve damage that cannot be restored by current therapies. Thus tests of new therapeutic approaches on experimental models with repeatable IOP increase are still required.
IOP in experimental models of glaucoma offers the chance to test the efficacy of new compounds in a controlled and reliable system. Thanks to the advanced equipment and knowledgeable staff, AF can offer highly controlled models of experimental Glaucoma in rats and mice. The retinal damage is evaluated and quantified using standard parameters such as the histological evaluation of the Retinal Ganglion Cells (RGCs), loss or modulation of specific markers of cellular survival or cell death.

 
Nonarteritic Anterior Ischemic Optic Neuropathy (NAION) 
AF offers a fully assessed rat model of NAION obtained by laser-mediated activation of an intravascular dye, with subsequent endothelial stress and vessel damage. The lesion induced on the anterior optic nerve is indistinguishable from those observed in the clinic, making this model an irreplaceable tool for efficacy evaluation of new drug candidates. The standard histological evaluation used to define and quantify the severity of the neuropathy is flanked by the measurement of the blood flow at the level of the optic nerve through the use of a Photo-acoustic Laser Doppler imaging system (VEVO 2100, Visualsonics). As for all models provided by the Biogem Service, efficacy studies can be tailored to match specific requirements.

Retinitis Pigmentosa
Retinitis pigmentosa is the most common cause of retinal degeneration. Visual impairment is primarily due to loss of photoreceptors, which leads to subsequent damage of the retinal pigment epithelium and other layers of the retina. Changes in retinal vasculature are prominent clinical features, with attenuation of retinal vessels in early stages and fibrotic degeneration in later stages of the disease. AF offered a specific animal model for this disease represented by the Royal College of Surgeons (RCS/Kyo) rats with inherited retinal degeneration. The genetic defect in the mertk gene causes the inability of the rethinal epithelium cells to phagocytose shed photoreceptor outer segment.

Dry Eye (Keratoconjunctivitis Sicca, KCS)
Dry eye syndrome is a common tears and ocular surface multifactorial disease, described by changes in the ocular surface epithelia related to reduced tears quantity and ocular surface sensitivity, leading to inflammatory reaction. Managing the eye inflammation proved helpful to patients with dry eye disease and current treatment is based on the use of topically applied artificial tear products/lubricants, tear retention management, stimulation of tear secretion and using anti-inflammatory drugs.

IBD inflammatory bowel diseases models (ulcerative colitis, Crohn's disease)
Inflammatory bowel diseases (IBD) are chronic inflammatory disorders including Ulcerative Colitis and Crohn's disease, mediated by genetic, immune, and environmental factors.
The therapeutic approach to this pathology is still not well addressed, and several novel drug candidates need to be developed. Chemically induced IBD animal models, available from the Biogem Service, can be used to dissect mechanism of action and to develop novel therapeutic strategies for IBD.

 
Acute colitis induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) model
The Acute Colitis model is obtained in outbred mice and rats through a single dose of 2,4,6 trinitrobenzene sulfonic acid (TNBS).
Sub Acute IBD models are obtained by a 7 days oral administration of Dextran Sulfate Sodium (DSS) in drinking water. These models can be useful to test both the preventive and/or curative effect of new compounds.
Clinical observations, gross and histopathology evaluation, gene expression profiling, and specific findings requested by the client can be used to characterize the severity of the lesions and to define the efficacy of the test compounds.

Diabetic Nephropathy (DN)

DN is a progressive kidney disease caused by longstanding Diabetes Mellitus (DM) and it is one of the most common DM complications. Furthermore, it is a prime reason for dialysis in many developed countries.
Although DN is a well-understood pathology, the currently available treatments can only slow down the progression of this disease: therefore, novel drug candidates are needed, especially in order to revert the symptoms of DN.

In our AF, obese-diabetic db/db mice are being used as a suitable and reproducible genetic model of DN, in order to develop novel therapeutic strategies for this pathology.

Rheumatoid Arthritis (RA)

RA is a long-term autoimmune disorder which primarily affects joints, and it is believed to be caused by genetic and environmental factors. It affects 5 to 50 per 100,000 people each year. The underlying mechanism of RA involves a strong autoimmune reaction against the joints. 
The principal therapeutic approach is symptomatic, with the goal of reducing pain and inflammation: analgesics, steroids, NSAIDs and disease-modifying antirheumatic drugs (DMARDs) are most frequently used.
To help the discovery of new pharmacological approaches to RA treatment, in our AF is currently being used a highly reproducible induced model of RA obtained through immunizations of DBA1/J mice with Collagen II, resulting in severe tarsal joint swelling and reddening starting from two weeks after the second immunization.

Cystitis

Cystitis is a common inflammatory pathology involving the urinary bladder, which has a prevalence of 6.2% to 11.2% in women and 2.3 to 4.6% in men. It is often caused by E. coli infection of the lower urinary tract, and represents the most frequent bacterial infection in women. 
With the aim of helping researchers in the discovery of novel therapeutic approaches to Cystitis, in our AF is available a suitable acute in-vivo model of this pathology, obtained through intraperitoneal injection of Cyclophosphamide (CYP) in female Sprague-Dawley rats. Furthermore, we offer the possibility to follow the development of cystitis through ecographic imaging using the VEVO 2100 equipped with LAZR (Visualsonics).

Type I and II Diabetes Mellitus (DM)

DM is a group of metabolic diseases characterized by high blood glucose levels over a prolonged period, whose incidence is increasing worldwide at an alarming rate. DM exists in two fundamental types: Type-I (Insulin-Dependent Diabetes Mellitus – IDDM) and Type-II (Non Insulin-Dependent DM – NIDDM), the latter accounting for 90-95% of all diabetes. Biogem Service offers a wide range of genetic and induced models of DM to help the discovery of novel therapeutic strategies for DM and DM and DM-related complications

Type-I Diabetes

a. Non-Obese Diabetic (NOD) mice

NOD mice represent the principal spontaneous experimental model for IDDM: in NOD mice, especially females, pathologic events start developing from the third week of age with the presentation of pancreatic islet antigens into the pancreatic lymphnodes, leading to insulitis within 15 weeks. These events trigger a strong autoimmune response against the pancreatic islets which leads to frank diabetes (fasting blood glucose ≥ 250 mg/dL) within 18 and 20 weeks of age.

b. Streptozotocin (STZ)-Induced Models

Single or multiple injections of STZ in mice or rats represent a widely used induced experimental model for IDDM: in this model, DM is induced in rats or mice through single or multiple intraperitoneal injections of STZ dissolved in citrate buffer. STZ is a cytotoxic agent which is highly selective for pancreatic β-cells, leading to pancreatic islets destruction and to the development of DM within 48 hours, with fasting blood glucose higher than 500 mg/dL.

Type-II Diabetes

a. Diet Induced Obesity (DIO) Models

DIO animals represent a highly useful induced model for studying NIDDM, obesity and hypercholesterolemia. In our AF, male C57 BL/6 mice or Sprague-Dawley (SD) rats are fed for 4-8 weeks with a High Fat Diet (HFD, containing more than 60% of fat) to induce obesity and obesity-related NIDDM and hypercholesterolemia.

b. Obese-Diabetic db/db mice

The db/db spontaneous mutant mouse model, carrying a homozygous loss of function mutation in the gene encoding for leptin receptor on a C57 BL/6 background, is a highly suitable model for studying NIDDM. The pathologic events start at 10 to 14 days of age with elevation of plasma insulin leading to frank diabetes at four to eight weeks of age. This model is characterized by obesity, polyphagia, polydipsia, polyuria and insulin resistance.

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.

List of Services

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

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.

Technical and veterinarian assistance for defining experimental protocols in order to monitor and keep animal welfare

Housing and management of mouse colonies according to the SPF standard on the basis of FELASA guidelines