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Deutsches Krebsforschungszentrum

1. Center for Preclinical Research

Cancer represents a devastating disease. The mission of the DKFZ is research on the molecular
mechanisms of carcinogenesis and the improvement and development of cancer therapies. Scientists at
the DKFZ work whenever possible with cell cultures or try to answer scientific questions by computer
simulation (Replacement). In many cases, due to the complexity of tumorigenesis the significance of
alternative in vitro or in silico systems is limited so that animal experiments are indispensable. These are
restricted to the necessary dimension (Reduction); animals are treated with respect and with the most
advanced techniques (Refinement). For their experiments, DKFZ scientists preferably use rodents, in rare
cases also amphibians. To mimic the development of a distinct cancer in humans, cancer researchers
employ various tumor models; genetically manipulated mice with a tissue-specific gain or loss of one or
several genes serve to evaluate the functional role of such genetic factors in cancer development.

To some extent, the mechanisms of tumor development are analysed in mice that have been treated with
carcinogenic agents. To study the characteristics of human tumor cells, mice with a compromised
immune system are often used, since they tolerate the transplantation of cells from other species and
allow their growth.

The Staff of the DKFZ Center for Preclinical Research supports scientists who are in need of animal
organs or who experiment with animals that were bred in-house or selectively purchased. A team of
clinical veterinarians and biologists advises and assists researchers in the design and realization of their
experiments. Managers of the ZTL train technicians and caretakers and provide technical courses for
scientific personnel. By these means, it is assured that all animals are kept according to national and
international animal protection laws. In addition, the welfare of the animals is ensured by maintaining
them under the highest level of hygiene. In the Laboratory of Microbiological Diagnostics, the health
status of the animals is regularly checked. The Transgenic Service supports scientists at DKFZ in the
generation of genetically modified mouse lines that serve for investigation of distinct human tumor
diseases. In the Cryopreservation Unit, the most important mouse strains are preserved for further studies
by collecting spermatozoa or early-stage embryos from mutant mice and subsequent freezing in liquid
nitrogen. In addition, a database of all mouse mutants available in-house is provided. The Tumor Models
Unit realizes cancer studies in experimental animals by application of carcinogenic agents and by
transplantation of tumor cells into immune-deficient mice. Moreover, diet and therapy studies are carried
out.

1.1. Tumor Models

Malignant tumors are complex tissues which arise from normal cells in a protracted multi-step process.
Driving forces of tumor development are the progressive accumulation of genetic and epigenetic DNA
alterations. As a consequence, such changes disturb cellular signaling pathways and regulatory circuits in
affected cells conferring growth advantages, landscaping the micro-environment of the tumor cells, and
supporting escape from immunosurveillance. Concomitantly, new blood vessels are formed,
inflammatory cells are recruited, and fibroblasts are activated in the surrounding tumor stroma. The
complex interactions between tumor cells and vessel, immune, or stroma cells are not yet traceable in an
authentic manner in cell cultures in vitro. Therefore, in vivo mouse models of cancer including
genetically engineered mouse lines and hetero-grafted mice are indispensable tools for the exploration of
the molecular and cellular fundamentals of tumor growth as well as translational applications in cancer
research.

The Core Facility Tumor Models offers expertise, specific technical equipment, and resources to
efficiently and successfully organize the design, realization, and data interpretation of animal experiments
in approved in vivo cancer-related models, thereby using state-of-the-art-protocols that comply with legal
requirements.

Our current offer of service comprises:

1. Inflammation tests
2. Multi-stage carcinogenesis tests in mouse skin
3. Heterograft tumor models
4. COX-2 tumor models
5. MITO database for models in Translational Oncology

The tasks will be met either in a full or in an assisted service. This implies a training of participating
experimenter, particularly in the application of carcinogens and irritating agents. The range of tumor
models will be adjusted to changing scientific needs.

1.2. Central Animal Laboratory

Members of the Central Animal Laboratory breed and house animals for all Research Groups of the
DKFZ. In addition to rats (Rattus norvegicus), guinea pigs (Cavia porcellus), mastomys (Mastomys
coucha), and amphibians (Xenopus species), genetically manipulated mice (Mus musculus), in particular
play a crucial role in cancer research.

A team of Clinical Veterinarians specialized in laboratory animal science is heading the facility. Qualified
Animal Technicians working in a team take care of specific animal colonies. Advanced education courses
and enhanced in-house training are the basis for optimal qualified Animal Technicians. An intensive and
constructive cooperation between Veterinarians, Animal Technicians and Scientists ensure not only the
legal requirements with regard to housing, space, temperature, humidity and enrichment etc. but also
ensures minimal stress for animals in experimental conditions. For documentation of breeding, a
specialized software program is installed to ensure adequate transparency for all concerned.

Not only do the animal welfare regulations require that animals should be free from disease, it is also an
important prerequisite for gaining meaningful experimental results. The Specific Pathogen-Free status
(SPF) is achieved by housing the animals in special barriers or in individually ventilated cages (IVC).
Through close cooperation with Personnel from the Unit Microbiological Diagnostics, the animals are
closely monitored. With these control standards, animals with an optimal microbiological quality are
provided for research. With regard to importation of animals, there will always be a need for cleaning up
mouse strains that are contaminated with undesirable pathogenic microorganisms. This cleaning up
process is performed through an embryo transfer system; mouse embryos that are only a few days old are
removed from the contaminated donor mother and then transplanted into an SPF hygiene status recipient
mother. The mother and her offspring are then maintained in a positive pressure isolator. Following
intensive microbiological examination to establish the health status, the offspring can then be transferred
to the SPF area of choice.

To mimic genetic changes in human tumors, in cancer research mouse models have been established in
inbred strains of a high standardized background; these mouse models carry appropriately mutated genes
or gene combinations known as transgene/transgenic or knockout allele(s). In order to check the
authenticity of inbred strains, a program for systematic analysis of a panel of single nucleotide
polymorphisms (SNPs) has been introduced.

1.3. Microbiological Diagnostics

Utilizing a minimum number of experimental animals, meaningful scientific results can only be obtained
if the animals are free of pathogens that may falsify experimental results. Microbiologically standardized
animals are a prerequisite for restricting animal experiments to the “inevitable amount” required by law.
It is the task of the Microbiological Diagnostics laboratory to prevent the introduction of harmful
microorganisms into laboratory animal care at the Center and to make sure that the animals kept at the
Central Animal Laboratory are free of infections or parasites. Before use in experiments, biological
materials such as cells, tumor samples, and serums are also screened for contaminations by bacteria,
fungi, and viruses.

Further services provided:

 Microbiological counseling
 Providing reference organisms (especially rodent-relevant bacteria, viruses)
 Providing polyclonal antibodies against microorganisms of mice and rats
 Detection of antibodies against microorganisms that are pathogenic in rodents
 Compiling results of microbiological studies for publications, health certificates

1.4. Transgenic Service

The Transgenic Service supports scientists at the DKFZ in the generation of genetically modified
(transgenic) mouse lines. This procedure involves inserting or deleting DNA sequences into the animals’
genetic material. Depending on the scientific question an inserted gene can be translated into a
biologically active protein, or a specific gene of the recipient animal can be modified or silenced.

Three main methods are used for generating genetically modified mice (e.g., as model systems for
investigating human diseases):

DNA microinjection: Foreign DNA is injected into fertilized mouse egg cells (zygotes) which are borne
by foster mice. Part of the newborn mice will have integrated the foreign DNA into their own
chromosomes.

CRISPR/Cas (endonuclease mediated genome editing): RNA (gRNA and Cas9 mRNA) and DNA
(template) are injected into fertilized mouse egg cells (zygotes) which are born by foster mice. Part of the
newborn mice will have integrated a specific genetic modification into the mouse genome.

ES cell microinjection: DNA is introduced into embryonic stem cells (ES cells) of mice and these are then
injected into early mouse embryos (blastocysts). The cells containing the foreign DNA will be integrated
into the embryo. Thus it is possible to generate mice with mutations in specific genes (such as “knockout”
mice). The effects observed in mice with such knockout mutations provide insight on the biological
function normally performed by the silenced gene.
2. Electron Microscopy

Electron microscopy allows to image biological samples down to macromolecular resolution.

Intrinsically, such structural detail is difficult to obtain demanding expertise in sample preparation and
instrument operation.

The central unit EM is dedicated to serve researchers of the DKFZ to perform experiments which use
transmission electron microscopy. We offer full service in EM-preparation and -imaging, notably
conventional resin-EM for cells and tissues and negative staining for particle preparations (viruses,
macromolecular complexes).

3. Light Microscopy

The Light Microscopy unit offers a variety of advanced imaging-related services for all in-house groups
and their collaboration partners at several locations on the campus. We provide consultation, hands-on
training, technical support and open access (24/7) to cutting-edge instrumentation and a broad range of
applications. An integrated workflow guides the user all the way through the required process steps,
starting from sample preparation, through data acquisition, up to data analysis and digital image
processing.

A large number of classical fully automated widefield and confocal laser-scanning microscopes is
supplemented by non-standard imaging systems for super-resolution microscopy (own development),
slide-scanning, high-content live-cell microscopy, spinning-disk microscopy, multiphoton microscopy,
optical projection tomography, etc.

Our sample preparation section comprises all the necessary equipment for cell culture, paraffin and
cryosectioning, histological staining, as well as instrumentation for micromanipulation techniques such as
laser capture microdissection and microinjection. The users can choose between assisted support and full
service. Our fluorescent marker repository provides access to a range of (genetic) markers for
morphological and physiological determination.

Big data is becoming a more and more important subject in light microscopy applications. We
accommodate the increasing demand for automated digital image processing by providing high-end
working stations, appropriate software (e.g. ImageJ/Fiji, Amira, Imaris, ZEN, LAS, Matlab, GraphPad
PRISM, SigmaPlot), as well as professional support for algorithm and macro development.
Besides the daily-based individual introductions at the devices, we offer open workshops in collaboration
with our external partners as well as topic-specific courses where the users familiarise themselves with
the theoretical background and the appropriate and autonomous usage of the complex devices and
applications such as multidimensional image acquisition, ratio imaging, FRAP, FRET, FCCS, 3D
reconstruction and deconvolution, colocalisation, spectral unmixing, etc.

4. Flow Cytometry (FACS ®)

Thanks to continous technical development and the use of novel fluorescent dyes, the field of application
of flow cytometry has steadily expanded in recent years. Originally developed as a method in
immunology and haematology (routine diagnostics), flow cytometry is increasingly used in other
biomedical research areas and in basic medical research. Especially in single cell analysis (e.g. genome
and transcriptome analysis), flow cytometry cell sorting plays an important role for obtaining and
processing the sample material.

FACS® (Fluorescence Activated Cell Sorting) analysis is based on a (stained) single cell suspension (or
other soluble particles), which individually pass through a focused laser beam and the characteristic
scattered and fluorescent light generated is detected separately. With this method, relatively large cell
counts can be analysed in a comparatively short time (up to 30,000 cells per second). The number of
parameters per cell that can be analysed increases with the number of lasers and fluorescent dyes used.
With our cell sorting devices at the DKFZ we can detect twenty parameters at the single cell level
simultaneously with the above-mentioned flow rate and sort four subpopulations in parallel.

For the staining of cells, fluorescence dye-labelled antibodies are generally used, which specifically
recognize and label structures on the cells. By suitable selection and composition of the individual
markers with different fluorescent dyes, several dozens of subpopulations can be distinguished in one
panel.

The Cytometry Core Facility at the DKFZ is a central service facility that advises and supports scientists
in the planning, execution and evaluation of flow cytometry analyses and cell sorting. Modern
instruments (analytical instruments and cell sorters) as well as various software are available for this
purpose. In addition, the flow cytometry core facility is involved in the training of DKFZ staff and
doctoral students. The courses on offer can be viewed via the department for advanced training. Course
materials are made available via the DKFZ's internal eLearning portal.
After training (participation of our lectures and practical introduction and/ or training by the core staff)
and acknowledgment of our user-guidelines, the use of the analysis equipment is directly done by the
users. The cell sorting devices are operated with the support of our core members. Registered users fill
out an "Initial Sort Discussion Sheet" and reserve a sort time slot online, which must be confirmed by the
core facility members.

DKFZ staff can find further information on our range of services and available equipment on the Intranet
under Scientific Services - Core Facilities – Imaging and Cytometry - Flow Cytometry

5.Genomics and Proteomics Core Facility

5.1.High Throughput Sequencing Unit

The high throughput sequencing unit provides highly parallel DNA sequencing services based on the
Illumina HiSeq 2000 / 2500, Hiseq 4000, HiSeq X, MiSeq, NextSeq and NovaSeq technologies as well as
on the PacBio Sequel.

Applications

 DNA Sequencing

o Whole Genome Sequencing

o Targeted Resequencing (e.g. Exome Sequencing)
o De-Novo Sequencing
 RNA-Sequencing

o mRNA stranded
o smallRNA
o UltraLowRNA
 Bisulfite Sequencing »...

o Analysis of DNA methylation patterns via Whole Genome

 ChipSeq  »...
o Generation of precise DNA-protein interaction maps
o Detection of transcription factor binding sites

Our services
  Assistance in experimental design
 Sample preparation starting from customer prepared DNA/RNA
 Sequence analysis on Illumina:
o HiSeq 2000 (V4: 125 bp Paired End or 50 bp Single Read)
o HiSeq 2500 (100 bp or 150 bp Paired End Rapid)
o HiSeq 4000 (100 bp Paired End)
o HiSeq X (150 bp Paired End)
o MiSeq (V2: 150 bp or 250 bp Paired End or 50 bp Single Read; V3: 75bp or 300 bp
Paired End or 150 bp Single Read)
o NextSeq 500 (MidOutput: 75bp or 150bp Paired End; HighOutput: 75bp Single Read or
75bp or 150bp Paired End)
o NovaSeq 6000 (50bp and 100bp Paired End on S1 and S2 Flowcell; 150bp Paired End on
S1, S2 and S4 Flowcell)
 Sequence analysis on PacBio Sequel
 Provision of the full sequence data
 Basic bioinformatics support for the analysis of resulting sequence data

5.2. Expression Profiling Service

The Microarray Unit is equipped with all major microarray expression platforms. Gene expression
profiling is offered comprehensively, providing Affymetrix (cartridge based) platform with all its
respective mRNA analysis opportunities. Agilent array platforms are mostly used for gene expression
analysis in specific model systems (e.g. E. coli, Drosophila melongaster).

YOU supply

 Completion of sample sheet

 High-quality intact RNA (assess RNA quantity and integrity)
 DNA Sample Concentration and Volume Requirements:

  Affymetrix Agilent
Chip-Format 1 4/8
(No.Samples/Chip) (depending on Chiptype)
Min. Concentration/sample 50 ng/µl 50 ng/µl
Total RNA/sample >500 ng >500 ng
Volume >8 µl >8 µl
 
For lower RNA amounts please inquire.

 All samples should be treated with DNase

 Samples must be non-pathogenic and non-infectious (S1-Condition).
 RNA samples must be provided in a nuclease-free 1.5ml-tube and on dry-ice

 Please be aware: NO BIOLOGICAL REPLICATES = NO STATISTICS

Please consult the pricelist for detailed cost information.

 We perform

 Assistance with Experimental Design

 Incoming QC for quality and concentration of all samples (Nanodrop ND-1000, Agilent 2100
Bioanalyzer)
 Labeling and hybridization to the microarrays you request
 Monitoring the quality at all steps
 Basic Data Analysis

5.3. miRNA Profiling

MicroRNAs (miRNAs) are evolutionarily conserved, small, noncoding RNA molecules that negatively
regulate gene expression at the level of translation in a variety of eukaryotic organisms. Although the first
miRNA was described in 1993, but only recently the breadth and diversity of this gene class been
uncovered. Vertebrate genomes encode as many as 1000 unique miRNAs, which are predicted to regulate
expression of at least 30% of genes.

5.4.Methylation Analysis

Epigenetics describes the study of heritable changes in gene function that occur without a change in the
nuclear DNA sequence. In addition to RNA-associated silencing and histone modification, a major
epigenetic mechanism in higher eukaryotes is DNA methylation.

5.5.Genotyping/CNV
Genotyping provides a measurement of the genetic variation between members of a species. Single
nucleotide polymorphisms (SNP) are the most common type of genetic variation. A SNP is a single base
pair mutation at a specific locus, usually consisting of two alleles.

6. Small Animal Imaging Center

The Small Animal Imaging Center supports users in the application of numerous radiological methods
such as ultrasound, optical imaging, µPET, µCT, µSPECT and MRT measurements to collect and
quantify morphological and functional data in vivo.

Multimodal Imaging (in vivo)

•  creation of illustrations
•  tumor staging
•  Screening for metastases
•  Intraindividual monitoring of therapy progress

Measurement of quantitative data (in vivo)

 blood volume
 vascular permeability
 perfusion
 oxygen saturation
 bone density (HU)
 volumetric measurement
 morphology
 metabolic activity
 pharmacokinetics

Further services

 Project planning of animal studies

 feasibility studies
 Evaluation of raw data
 scientific evaluation of the data
  data storage
 Support writing applications and publications

Small Animal Imaging Center

Modern non-invasive small animal imaging for comprehensive evaluation of pathologies and therapy
nitoring in vivo.

Under the direction of Dr. Manfred Jugold, the Core Facility Small Animal Imaging offers a
comprehensive service for the non-invasive characterization of small animal pathologies for DKFZ
internal groups only.

The radiological possibilities in today’s small animal imaging techniques are able to obtain a multitude of
significant data and meaningful images from living animals. By means of most animal-friendly
procedures, entire therapy processes can be documented in vivo during the course of the study.

Through the modern methods of non-invasive small animal imaging it is often possible to strongly reduce
the number of animals needed to achieve a study goal. At the same time, the significance of the results
can be maintained or even increased and additional parameters can be examined. In this way, decisive
contributions can be made to research on animal models, from simple documentation and the recording of
pathologies, through therapy monitoring to the measurement of quantitative and functional tissue
parameters. Furthermore, results, e. g. for publications or patent applications can be enhanced by
meaningful images.

Compared to conventional invasive or approximate methods, small animal imaging offers decisive
advantages:

 Recording of quantitative volumetric data

 Monitoring of intraindividual therapy courses
 Recording of functional tissue parameters
 Increasing the significance of the results
 Imaging of finest morphologic structures
 Rationalisation of animal numbers

Furthermore, we offer support in the design of animal studies, the selection of suitable modalities, the
processing of the raw data,as well as the evaluation/interpretation of the results and the publication of the
study.

Available modalities and equipment: MRI: Icon, Bruker

 1Tesla small animal scanner

Three-modality small animal scanner: Inveon, Siemens

 μCT
 μPET
 μSPECT

Photoacoustic Imaging: Fujifilm, VisualSonics

 LAZR-Photoacoustic-Imaging-System
Wavelength range: 680 - 970 nm and 1200 - 2000 nm

Optical imaging: PerkinElmer

 IVIS Lumina III, In Vivo Imaging System Bioluminescence Imaging Fluorescence Imaging

Ultrasound: Fujifilm, VisualSonics

 Vevo 3100™ Transducer mouse 13-24 MHz Transducer rat 22-55 MHz

7. Metabolomics Core Technology Platform

Depending on the requirements of your project, we offer tailor-made solutions as well as standardized
Metabolomics assays, e.g.:

 Comparative metabolite screening of central carbon and nitrogen metabolism

 Single compound analysis
 Targeted metabolite profiling

Examples of established targeted analyses of diverse metabolite classes:

 Carbohydrates / Sugars
  Citrate cycle compounds (Dicarboxylic organic acids, e.g. Malate, Fumarate, Citrate)
 α-Ketoacids (e.g. Pyruvate, Ketoglutarate)
 Amino acids
 Polyamines
 Nucleotides (e.g. AMP, ADP, ATP, GTP, CTP, UTP)
 Thiols (Glutathione - GSH/GSSG - and Cysteine)
 Polyphenols (e.g. Phytoestrogenes, Flavonols)

A detailed list of all compounds determined so far is available upon request.

For analyses of specific metabolites or biomarkers, please contact the MCTP.
Our service includes extraction, separation,detection of metabolites, and basic bio-informatic data
analysis. Results are usually provided as Excel spreadsheet.